Difference between revisions of "Team:NTU-LIHPAO-Taiwan/Notebook"

Revision as of 21:49, 16 September 2015

NTU-LIHPAO-Taiwan

Protocols

Preparation of Competent Cells (E.coli)

Materials and Reagents :

Escherichia coli DH5α
LB broth
TB buffer
DMSO
Liquid nitrogen

Equipment :

Ice
Shaker
Spectrometer
Centrifuge

Procedure :

Inoculate 10μL Escherichia coli DH5α into 10mL LB broth (1:1000)
Grow for 12-16 hours at 37℃ with shaking
Inoculate 500μL Escherichia coli DH5α into 50mL LB broth (1:100)
Grow for 2 hours at 37℃ with shaking to O.D.₆₀₀=0.4-0.6
Split the cell into two Falcon tubes, both contain 25 mL Escherichia coli DH5α
Centrifuge at 4℃, 3000rpm(800G) for 10 minutes
Discard the supernatant
Resuspend the cell pellet by gently adding 8.5mL TB buffer (1/3 V)
On ice for 10 minutes
Centrifuge at 4℃, 3000rpm(800G) for 10 minutes
Discard the supernatant using pipetman
Resuspend the cell pellet by gently adding 2mL TB buffer (1/12.5 V)
Add 150μL DMSO (7%)
On ice for 10 minutes
Transfer the cell to new eppendorfs with 60μL per tube
Freeze the cell in liquid nitrogen
Store the cell at -80℃

DNA Dissolution

Materials and Reagents :

DNA (BioBricks/synthesized)
ddH₂O

Procedure :

Add 10μL ddH₂O
Wait for 1 minute until the DNA dissolve
Pipet several times and transfer the DNA to an eppendorf

Agarose Gel Electrophoresis

Materials and Reagents :

Agarose
1X TAE
Tracking dye
Marker (1kb/100bp)
EtBr
ddH₂O

Equipment :

Gel tray
Well comb
Electrophoresis tank
UV detector

Procedure :

Casting an Agarose Gel (m/v = 1.0% or 1.5%)

Measure out appropriate mass of agarose powder into a serum bottle
Add appropriate volume of 1X TAE
Let agarose solution cool down to acceptable temperature for bare hands
Pour the solution into a gel tray with the well comb in place, and push the bubbles away with a pipette tip
Let sit at room temperature for 30 minutes, until it has completely solidified

Loading Samples and Running the Gel

Place the gel as well as its tray into the electrophoresis tank containing 1X TAE
Mix the samples with tracking dye (1/10 V) sufficiently
Load the samples into the each well
Load marker (usually in the first and last lane)
Set an appropriate voltage (full/half) and run the gel for 15-20 minutes

Imaging the Gel

Put the gel into a container filled with 1X TAE and EtBr, staining for 5 minutes
Replace EtBr solution with water and destain for 3 minutes
Put the gel in an UV detector and record the result

Gel Extraction

Materials and Reagents :

Gel/PCR buffer
W1 buffer
Wash buffer
ddH₂O

Equipment :

Cutter knife
Eppendorf
Vortex mixer
Dry bath incubator
DF Column & Collection tube
Mini Centrifuge
Microcentrifuge

Procedure :

Gel Dissociation

Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice (<300μL)
Transfer the gel slice to an eppendorf
Add 500μL Gel/PCR buffer to the sample and mix by vortex
Incubate at 60℃ for 10-15 minutes to ensure the gel slice has been completely dissolved (invert the tube every 2-3 minutes)
Cool the dissolved sample mixture to room temperature

DNA Binding

Place the DF Column in a 2mL Collection tube
Transfer 800μL of the sample mixture to the DF Column
Discard the flow-through and place the DF Column back in the Collection tube

Wash

Add 400μL W1 buffer into the DF Column
Spin down for approximately 20 seconds
Discard the flow-through and place the DF Column back in the Collection tube
Add 600μL wash buffer (contains ethanol) into the DF Column
Let stand for 1 minute at room temperature
Spin down for approximately 30 seconds
Discard the flow-through and place the DF Column back in the Collection tube
Centrifuge at 12000 rpm for 3 minutes to dry the column matrix
Discard the flow-through

DNA Elution

Transfer the dried DF Column to a new eppendorf (cap cut)
Add 30μL of 37℃ ddH2O into the center of the column matrix
Let stand for at least 2 minutes to ensure that ddH2O is completely absorbed
Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA
Re-add the subnatant into the center of the column matrix
Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA

DNA Ligation

Materials and Reagents :

DNA (insert & vector)
Ligation high ver.2

Equipment :

Cooling dry bath incubator

Procedure :

Table

Components Volume (μL)

Insert x

Vector y

Ligation High 1

Total 7

Molar ratio: insert/vector = 3/1
x + y = 6
Gently mix the solution by pipetting up and down
Incubate
1. at 16℃ for 2 hours, or
2. at 37℃ for 1 hour, or
3. at 4℃ overnight
Proceed with bacterial transformation

Plasmid DNA Extraction Using Alkaline Lysis Method (E. coli)

Materials and Reagents :

Escherichia coli DH5α (with plasmid)
LB broth
Resuspension solution (MPI, with RNAase)
Lysis solution (MPII)
Neutralizing solution (MPIII)
Isopropanol
70% ethanol
ddH₂O

Equipment :

Shaker
Centrifuge
Vortex mixer

Procedure :

Grow bacteria in LB broth with appropriate antibiotics at 37℃ overnight with shaking
Transfer 1.5mL culture to an eppendorf
Centrifuge at 4℃, 5000rpm for 5 minutes
Discard the supernatant
Add 150μL of resuspension solution (MPI, with RNAase) into each tube, pipet several times, and vortex to completely resuspend the cell pellet
Add 150μL of lysis solution (MPII), gently invert the tubes about 20 times, and then let the sample mixture stand at room temperature for 2 minutes
Add 150μL of neutralizing solution (MPIII) and mix by inverting the tubes about 20 times. Bacterial chromosomal DNA and proteins can be seen as white precipitates
Centrifuge at 4℃, 15000rpm for 15 minutes
Carefully transfer 400μL of the supernatant to a new eppendorf
Add 400μL (same volume as the supernatant) isopropanol, and shake up the tubes as vigorously as one can
Centrifuge at 4℃, 15000rpm for 15 minutes
Discard the supernatant
Add 200μL of 70% ethanol to wash out the salts
Centrifuge at 4℃, 15000rpm for 5 minutes
Discard the supernatant and remove the remains as much as possible with pipetman
Air dry for about 30 minutes
Resuspend the DNA pellet with 20μL ddH₂O

Transformation (E. coli)

Materials and Reagents :

Competent cell (Escherichia coli DH5α)
LB plate (Amp+/CP+)

Equipment :

-80℃ refrigerator
Ice
37℃ incubator
Super optimal broth (SOB) (37℃)
Shaker

Procedure :

Take out the competent cells from -80℃ refrigerator
Thaw the cells on ice for 5 minutes
Add 1μL plasmid DNA into the competent cells, and mix gently by pipetting
On ice for 10 minutes
Heat shock at 37℃ for 3 minutes
On ice for 2 minutes
Add 150μL 37℃ SOB into the mixture
Place the tube at 37℃ with shaking for 1 hour(Amp+)/4 hours(CP+) respectively
Spread the cells onto the LB plates (Amp+/ CP+)
Incubate the plates at 37℃ with shaking for 12-16 hours

Protein Extraction

Materials and Reagents :

plasmid/ BL21 competent cell
LB plate
LB broth
lysis buffer

Equipment :

15/ 50ml conial tube
Shaking incubator
Erlenmeyer flask
Spectrophotometer
Centrifuge
Sonicator
Eppendorf

Procedure :

Transform plamid into BL21 (see transformation)
Pick up a single colony and incubate into 2ml LB/CP medium, then agitate them in a shaking incubator at 37℃ overnight
Pour the culture into 100ml LB/CP medium, then agitate until it reach A₆₀₀ of 0.6
Pour the culture into 50ml conical tube, and centrifuge at 8000rpm for 10 min
Store the supernate and the pellets in the centrifuge tubes in a -80℃ freezer
Use 6ml lysis buffer(NP-10) to resuspend the cell pellet and transfer into a 15ml centrifuge tube
Lyse the cell with sonication
Centrifuge 15ml conial tube at 9000rpm for 10 min
Store the supernate as coarse extract

SDS-PAGE

Materials and Reagents :

Solution A (acrylamide/ bis-acrylamide (29:1) 30% solution)
Solution B (Tris (Tris base, 1.5 M) 90.8 gm; TEMED 1.8 ml; pH = 8.8/ 500ml)
Solution C (Tris (Tris base, 0.5 M) 6.0 gm; TEMED 0.4 ml; pH = 6.7/ 100ml)
10% SDS (1kb/100bp)
ddH₂O
APS
Running buffer (1M Tris 12.5 ml; glysine 7.7g/ 500ml)
Sample buffer(5X) (Tris base, 125mM X5; EDTA2•Na, 2mM X 5; SDS(2% X2); β-mercaptoethanol, 5% X5)
Protein marker

Equipment :

Gel Caster
Accessories for each gel
(1) One glass plate
(2) One aluminum notched plate
(3) Two 0.75-mm thick spacers
(4) One 0.75-mm thick 10-well comb
(5) One well-locating decal
Power supply

Procedure :

Assemble one glass plate and one alumimum plate with two spacers for each gel module
Prepare the SDS gel solution (see the table below). Two 0.75mm mini gels need approximately 10ml separation gel solution and 5ml stacking solution. Add APS solution at the final step
Mix the separation gel solution, and add the solution into the gel module until reaching the designated level. Then add isopropanol on the top of the gel. It takes about 40 min to complete the polymerization step
Remove the isopropanol by using a small strip of filter paper. Fill up the stacking gel solution until reaching the top. Insert comb into the gel solution as soon as possible. It takes 10 min to complete the polymerization step
Remove the comb and set the gel in the cell buffer dam. Pour the running buffer into the inner chamber and keep pouring after overflow until the buffer surface reaches the required level in the outer chamber
Use a micropipette to clean up every single well with the running buffer
Mix protein samples with 1/5 volumes of 5X sample buffer. Heat the mixture at 100oC for 10 min. Let it cool down to room temperature
Load protein sample and the protein marker into the well
Run the electrophoresis at 70V for 20 min, and then 150V for 55 min
Remove the two spacer and lift the glass plate, and remove the stacking gel and the remaining gel is ready for CBR staining and Western blot

(Unit: ml)	Separation gel						Stacking gel
Percentage	5%	7.5%	10%	12.5%	15%	20%	4%
Solution A	16.5	2.5	3.35	4.15	5	6.5	0.66
Solution B	2.5	2.5	2.5	2.5	2.5	2.5	-
Solution C	-	-	-	-	-	-	1.24
10% SDS	0.1	0.1	0.1	0.1	0.1	0.1	0.05
dH₂O	5.7	4.85	4	3.2	2.35	0.85	2.95
APS(10%)	0.05	0.05	0.05	0.05	0.05	0.05	0.1
Total Volume	10	10	10	10	10	10	5

Western Blot

Materials and Reagents :

Transfer buffer ( Tris 25mM; glycine 0.192M)
Methanol
10 X TBST (1M Tris pH =8.0; Tween 20, 50ml; NaCl 43.5g)

Equipment :

Semi-dry trnasfer cell
PVDF membrane
Nitrocellulose paper
Square Petri dish
Rotary shaker

Procedure :

Equilibrate the nitrocellulose paper and SDS-PAGE gel by soaking them with transfer buffer
Rinse the PVDF membrane with 100% methanol for a few second and equilibrate it with transfer buffer
Place the elements in the following order onto the semi-dry transfer cell from bottom to top: nitrocellulose paper, PVDF membrane, SDS-PAGE gel, nitrocellulose paper
Set up the current at 0.09A and run for 75 min

Immunostaining

Materials and Reagents :

3% milk
Antibody solution
Secondary antibody (anti-mouse HRP)
TBST
Western HRP substrate

Equipment :

Square petri dish
Rotary shaker
UVP Biospectrum

Procedure :

Put the membrane in the square petri dish block the membrane with the 3% milk/TBST at room temperature for 1hr
Discard the milk and replace it with antibody solution(mouse His-probe Antibody) at 4℃ overnight
Recycle the antibody solution. Wash the membrane with TBST three times, 5 min each
Soak membrane into anti-mouse HRP solution for 1 hr
Discard the anti-mouse HRP solution and wash it with TBST three times, 5 min each
Add HRP substrate on the membrane for 1 min
Soak the membrane into ddH₂O to remove the substrate
Observe the result with UVP Biospectrum

Thawing Caco-2 cells

Materials and Reagents :

1 ml frozen Caco-2 cells in cryovial
Cell culture medium (MEM (minimum essential medium) (GIBCOTM #41500-034)/2.2g NaHCO3/ 0.11 g sodium pyruvate/20% FBS/1% PSA)
70% ethanol
Trypan blue solution

Equipment :

Laminar flow hood
Auto pipette
Centrifuge
15 ml centrifuge tubes
10 cm dish
Hemocytometer
Optical microscope
Suction machine

Procedure :

Pre-warm medium in 37˚C water bath
Open a laminar flow hood
Clean materials, reagents and equipments before moving them into the hood
Thawing Caco-2 cells in 37˚C water bath and transfer the cells into a 15ml centrifuge tube
Add 9 ml cell culture medium very slowly
Centrifuge at 1000 rpm for 5 min
Decant the supernate and resuspend cells with 10 ml cell culture medium
Mix 10 ul cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer
Add all cell suspension to a 10 cm dish
Write date, cell line and passage number
Put the dish into incubator

Freezing Caco-2 cells

Materials and Reagents :

Caco-2 cell in 10 cm dish
Cell culture medium (MEM (minimum essential medium) (GIBCOTM #10370-021)/10% FBS/ 2 mM glutamine
1X PBS (phosphate buffer saline)
0.5% trypsin
Trypan blue solution
70% ethanol
Cell freezing media (50% FBS, 40% cell culture medium, 10% DMSO)

Equipment :

Laminar flow hood
Auto pipette
Centrifuge
15 ml centrifuge tubes
10 cm dish
Hemocytometer
Optical microscope
Suction machine
Cryovials
Freezing apparatus
-80˚C refrigerator
Liquid nitrogen tank

Procedure :

Pre-warm medium in 37˚C water bath
Open a laminar flow hood
Clean materials, reagents and equipments before moving them into the hood
Take a 10 cm dish with Caco-2 cell
Discard the medium with a suction machine
Wash the cells with 5 ml PBS
Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes
Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface
Transfer the cell suspension to a 15 ml centrifuge tube
Mix 10 μl cell suspension and 10 ul trypan blue solution and count total cell number with a hemocytometer
Add about 1x10⁶ cells per cryovial, and add cell freezing media
Freeze the cryovials in a controlled rate freezing apparatus, decreasing the temperature approximately 1°C per minute in a -80°C refrigerator for more than 3 hour, then move them into liquid nitrogen tank

Caco2 Cell passage

Materials and Reagents :

Caco-2 cell in 10 cm dish
Cell culture medium (MEM (minimum essential medium) (GIBCOTM #10370-021)/10% FBS/ 2 mM glutamine
1X PBS (phosphate buffer saline)
0.5% trypsin
Trypan blue solution
70% ethanol

Equipment :

Laminar flow hood
Auto pipette
Centrifuge
15 ml centrifuge tubes
10 cm dish
Hemocytometer
Optical microscope
Suction machine

Procedure :

Pre-warm medium in 37˚C water bath
Open a laminar flow hood
Clean materials, reagents and equipments before moving them into the hood
Take a 10 cm dish with Caco-2 cell
Discard the medium with a suction machine
Wash the cells with 5 ml PBS
Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes
Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface
Transfer the cell suspension to a 15 ml centrifuge tube
Mix 10 ul cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer
Transfer appropriate amounts of cell suspension to new dishes (1/4 X)
Write date, cell line and passage number
Put the dish into incubator

Seeding in transwell

Materials and Reagents :

Caco-2 cell in 10 cm dish
Cell culture medium (MEM (minimum essential medium) (GIBCOTM #41500-034)/2.2 g NaHCO3/ 0.11 g sodium pyruvate/20% FBS/1% PSA
1X PBS (phosphate buffer saline)
0.5% trypsin
Trypan blue solution
70% ethanol

Equipment :

Laminar flow hood
Auto pipette
Centrifuge
15 ml centrifuge tubes
10 cm dish
Hemocytometer
Optical microscope
Suction machine
6-well plate
Millicell plate insert (PIHP03050)

Procedure :

Open 6-well plate, add 1.5 mL medium to each large well (6 wells) which are going to be used
Add 2 ml PBS/well to other wells
Put transwell in larger well
Take a 10 cm dish with Caco-2 cell
Discard the medium with a suction machine
Wash the cells with 5 ml PBS
Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes
Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface
Transfer the cell suspension to a 15 ml centrifuge tube
Mix 10 ul cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer
Cell count: 70/ 2 * orginal volume *100 (*10⁴)= 35 X 10⁴ cell/mL (Seeding 42 x 10⁴ cell/well)
Write date, cell line and passage number
Put the dish into incubator

Measure TEER

Materials and Reagents :

Caco-2 cells seeded in transwell
70% ethanol

Equipment :

Laminar flow hood
Millicell ERS

Procedure :

Prepare laminar hood for experiment
To sterilize electrode of Millicell ERS, soak electrode in 70% alcohol for 15 min, and air dry for 15 sec
Measure TEER value of each well (n=3)

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@@ Line 801: / Line 806: @@
 		<ul class="main-Content">
 			<li>
-				<span class="title">Months</span>
+				<span class="title">Protocols</span>
 				<ul class="sub-Content">
-					<li><a href="#First1">July</a></li>
+					<li><a href="#First1">Preparation of Competent Cells (<i>E.coli</i>)</a></li>
-					<li><a href="#First2">August</a></li>
+					<li><a href="#First2">DNA Dissolution</a></li>
-					<li><a href="#First3">September</a></li>
+					<li><a href="#First3">Agarose Gel Electrophoresis</a></li>
+					<li><a href="#First4">Gel Extraction</a></li>
+					<li><a href="#First5">DNA Ligation</a></li>
+					<li><a href="#First6">Plasmid DNA Extraction Using Alkaline Lysis Method (<i>E.coli</i>)</a></li>
+					<li><a href="#First7">Transformation(<i>E.coli</i>)</a></li>
+					<li><a href="#First8">Protein Extraction</a></li>
+					<li><a href="#First9">SDS-PAGE</a></li>
+					<li><a href="#First10">Western Blot</a></li>
+					<li><a href="#First11">Immunostaining</a></li>
+					<li><a href="#First12">Thawing Caco-2 cells</a></li>
+					<li><a href="#First13">Freezing Caco-2 cells</a></li>
+					<li><a href="#First14">Caco2 Cell passage</a></li>
+					<li><a href="#First15">Seeding in transwell</a></li>
+					<li><a href="#First16">Measure TEER</a></li>
 				</ul>
 			</li>
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-					<div class="Container2" id="First1"><div class="Text2">7/4</div></div>
+				<div class="Text1" id="First1"><Red>Preparation of Competent Cells (<i>E.coli</i>)</Red></div>
+					<div class="Text2">
+<Green>Materials and Reagents :</Green>
+					</div>
 						<div class="Text3">
-<Green>Manufacture <i>CP<Super>+</Super></i> plate</Green>
+<ol class="part1">
+	<li><i>Escherichia coli</i> DH5α</li>
+	<li>LB broth</li>
+	<li>TB buffer</li>
+	<li>DMSO</li>
+	<li>Liquid nitrogen</li>
+</ol>
 						</div>
+					<div class="Text2">
+<Green>Equipment :</Green>
-					<div class="Container2"><div class="Text2">7/6</div></div>
+					</div>
 						<div class="Text3">
-<Red>Learn how to transform</Red>
+<ol class="part1">
+	<li>Ice</li>
+	<li>Shaker</li>
+	<li>Spectrometer</li>
+	<li>Centrifuge</li>
+</ol>
 						</div>
+					<div class="Text2">
+<Green>Procedure :</Green>
+					</div>
 						<div class="Text3">
-<Green>Suicide Part:</Green>
+<ol class="part2">
+	<li>Inoculate 10μL <i>Escherichia coli</i> DH5α into 10mL LB broth (1:1000)</li>
+	<li>Grow for 12-16 hours at 37℃ with shaking</li>
+	<li>Inoculate 500μL <i>Escherichia coli</i> DH5α into 50mL LB broth (1:100)</li>
+	<li>Grow for 2 hours at 37℃ with shaking to O.D.<Sub>600</Sub>=0.4-0.6</li>
+	<li>Split the cell into two Falcon tubes, both contain 25 mL <i>Escherichia coli</i> DH5α</li>
+	<li>Centrifuge at 4℃, 3000rpm(800G) for 10 minutes</li>
+	<li>Discard the supernatant</li>
+	<li>Resuspend the cell pellet by gently adding 8.5mL TB buffer (1/3 V)</li>
+	<li>On ice for 10 minutes</li>
+	<li>Centrifuge at 4℃, 3000rpm(800G) for 10 minutes</li>
+	<li>Discard the supernatant using pipetman</li>
+	<li>Resuspend the cell pellet by gently adding 2mL TB buffer (1/12.5 V)</li>
+	<li>Add 150μL DMSO (7%)</li>
+	<li>On ice for 10 minutes</li>
+	<li>Transfer the cell to new eppendorfs with 60μL per tube</li>
+	<li>Freeze the cell in liquid nitrogen</li>
+	<li>Store the cell at -80℃</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+				<div class="Text1" id="First2"><Red>DNA Dissolution</Red></div>
-Transformation of:
+					<div class="Text2">
-						</div>
+<Green>Materials and Reagents :</Green>
-						<div class="Text3_SubSub">
+					</div>
-[RBS] (BBa_B0034), [C2] (BBa_C0053)
-						</div>
-					<div class="Container2"><div class="Text2">7/7</div></div>
 						<div class="Text3">
-<Red>Learn how to manufacture competent cell</Red>
+<ol class="part1">
+	<li>DNA (BioBricks/synthesized)</li>
+	<li>ddH<Sub>2</Sub>O</li>
+</ol>
 						</div>
+					<div class="Text2">
+<Green>Procedure :</Green>
+					</div>
 						<div class="Text3">
-<Blue>Manufacture competent cell</Blue>
+<ol class="part2">
+	<li>Add 10μL ddH<Sub>2</Sub>O</li>
+	<li>Wait for 1 minute until the DNA dissolve</li>
+	<li>Pipet several times and transfer the DNA to an eppendorf</li>
+</ol>
 						</div>
+				<div class="Text1" id="First3"><Red>Agarose Gel Electrophoresis</Red></div>
+					<div class="Text2">
+<Green>Materials and Reagents :</Green>
+					</div>
 						<div class="Text3">
-<Green>PYY part:</Green>
+<ol class="part1">
-						</div>
+	<li>Agarose</li>
-						<div class="Text3_Sub">
+	<li>1X TAE</li>
-Transformation of:
+	<li>Tracking dye</li>
-						</div>
+	<li>Marker (1kb/100bp)</li>
-						<div class="Text3_SubSub">
+	<li>EtBr</li>
-[promoter] (BBa_J23100), [signal peptide] (BBa_K554002), [terminator] (BBa_B0015)
+	<li>ddH<Sub>2</Sub>O</li>
-						</div>
+</ol>
+						</div>
+					<div class="Text2">
+<Green>Equipment :</Green>
+					</div>
 						<div class="Text3">
-<Green>Suicide Part:</Green>
+<ol class="part1">
+	<li>Gel tray</li>
+	<li>Well comb</li>
+	<li>Electrophoresis tank</li>
+	<li>UV detector</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+					<div class="Text2">
-Transformation of:
+<Green>Procedure :</Green>
-						</div>
+					</div>
-						<div class="Text3_SubSub">
-[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806), [C1] (BBa_C0051)
-						</div>
-					<div class="Container2"><div class="Text2">7/8</div></div>
 						<div class="Text3">
-<Red>Learn how to extract the plasmid</Red>
+<ol class="part2">
+	<div class="Text_TitleUnderline">Casting an Agarose Gel (m/v = 1.0% or 1.5%)</div>
+	<li>Measure out appropriate mass of agarose powder into a serum bottle</li>
+	<li>Add appropriate volume of 1X TAE</li>
+        <li>Let agarose solution cool down to acceptable temperature for bare hands</li>
+        <li>Pour the solution into a gel tray with the well comb in place, and push the bubbles away with a pipette tip</li>
+        <li>Let sit at room temperature for 30 minutes, until it has completely solidified</li>
+	<div class="Text_TitleUnderline">Loading Samples and Running the Gel</div>
+        <li>Place the gel as well as its tray into the electrophoresis tank containing 1X TAE  </li>
+        (Make sure that the surface is higher than the top of the gel and not overflow)
+        <li>Mix the samples with tracking dye (1/10 V) sufficiently</li>
+        <li>Load the samples into the each well</li>
+        <li>Load marker (usually in the first and last lane)</li>
+        <li>Set an appropriate voltage (full/half) and run the gel for 15-20 minutes</li>
+        <div class="Text_TitleUnderline">Imaging the Gel</div>
+        <li>Put the gel into a container filled with 1X TAE and EtBr, staining for 5 minutes</li>
+        <li>Replace EtBr solution with water and destain for 3 minutes</li>
+        <li>Put the gel in an UV detector and record the result</li>
+</ol>
 						</div>
+				<div class="Text1" id="First4"><Red>Gel Extraction</Red></div>
+					<div class="Text2">
+<Green>Materials and Reagents :</Green>
+					</div>
 						<div class="Text3">
-<Red>Learn how to conduct the gel extraction and electrophoresis</Red>
+<ol class="part1">
-						</div>
+	<li>Gel/PCR buffer</li>
+	<li>W1 buffer</li>
+	<li>Wash buffer</li>
+	<li>ddH<Sub>2</Sub>O</li>
+</ol>
+						</div>
+					<div class="Text2">
+<Green>Equipment :</Green>
+					</div>
 						<div class="Text3">
-<Green>Suicide Part:</Green>
+<ol class="part1">
+	<li>Cutter knife</li>
+	<li>Eppendorf</li>
+	<li>Vortex mixer</li>
+	<li>Dry bath incubator</li>
+        <li>DF Column & Collection tube</li>
+        <li>Mini Centrifuge</li>
+        <li>Microcentrifuge</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+					<div class="Text2">
-Extract the plasmid:
+<Green>Procedure :</Green>
-						</div>
+					</div>
-						<div class="Text3_SubSub">
-[RBS] (BBa_B0034), [C2] (BBa_C0053)
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[RBS] cleave by EcoRI, [C2] cleave by EcoRI and PstI
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text3_SubSub">
-[RBS] ----- Result: Failed (Smeared band was too blurred)
-						</div>
-						<div class="Text3_SubSub">
-[C2] ----- Result: Succeeded
-						</div>
-						<div class="Text3_Sub">
-Transformation of:
-						</div>
-						<div class="Text3_SubSub">
-[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806) <b>again</b>
-						</div>
-					<div class="Container2"><div class="Text2">7/9</div></div>
 						<div class="Text3">
-<Green>Suicide Part:</Green>
+<ol class="part2">
-						</div>
+        <div class="Text_TitleUnderline">Gel Dissociation</div>
-						<div class="Text3_Sub">
+	<li>Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice (<300μL)</li>
-Extract the plasmid:
+	<li>Transfer the gel slice to an eppendorf</li>
-						</div>
+	<li>Add 500μL Gel/PCR buffer to the sample and mix by vortex</li>
-						<div class="Text3_SubSub">
+        <li>Incubate at 60℃ for 10-15 minutes to ensure the gel slice has been completely dissolved (invert the tube every 2-3 minutes)</li>
-[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)
+        <li>Cool the dissolved sample mixture to room temperature</li>
-						</div>
+        <div class="Text_TitleUnderline">DNA Binding</div>
-						<div class="Text3_Sub">
+        <li>Place the DF Column in a 2mL Collection tube</li>
-Gel electrophoresis:
+        <li>Transfer 800μL of the sample mixture to the DF Column</li>
-						</div>
+        <li>Discard the flow-through and place the DF Column back in the Collection tube</li>
-						<div class="Text3_SubSub">
+        (If the sample mixture is more than 800μL, repeat the DNA binding step)
-[RBS] ----- Result: Failed (Smeared band was too blurred)
+        <div class="Text_TitleUnderline">Wash</div>
-						</div>
+        <li>Add 400μL W1 buffer into the DF Column</li>
-						<div class="Text3_SubSub">
+        <li>Spin down for approximately 20 seconds</li>
-[C1] ----- Result: Succeeded
+        <li>Discard the flow-through and place the DF Column back in the Collection tube</li>
-						</div>
+        <li>Add 600μL wash buffer (contains ethanol) into the DF Column</li>
-						<div class="Text3_SubSub">
+        <li>Let stand for 1 minute at room temperature</li>
-[endolysin] ----- Result: Failed (Smeared band was too blurred)
+        <li>Spin down for approximately 30 seconds</li>
-						</div>
+        <li>Discard the flow-through and place the DF Column back in the Collection tube</li>
-						<div class="Text3">
+        <li>Centrifuge at 12000 rpm for 3 minutes to dry the column matrix</li>
-<Green>PYY part:</Green>
+        (Can be done twice)
-						</div>
+        <li>Discard the flow-through</li>
-						<div class="Text3_Sub">
+        <div class="Text_TitleUnderline">DNA Elution</div>
-Extract the plasmid:
+        <li>Transfer the dried DF Column to a new eppendorf (cap cut)</li>
-						</div>
+        <li>Add 30μL of 37℃ ddH2O into the center of the column matrix</li>
-						<div class="Text3_SubSub">
+        <li>Let stand for at least 2 minutes to ensure that ddH2O is completely absorbed</li>
-[signal peptide] (BBa_K554002), [terminator] (BBa_B0015)
+        <li>Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA</li>
+        <li>Re-add the subnatant into the center of the column matrix</li>
+        <li>Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+				<div class="Text1" id="First5"><Red>DNA Ligation</Red></div>
-Gel electrophoresis:
+					<div class="Text2">
-						</div>
+<Green>Materials and Reagents :</Green>
-						<div class="Text3_SubSub">
+					</div>
-[signal peptide] ----- Result: Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[terminator] ----- Result: Succeeded
-						</div>
-					<div class="Container2"><div class="Text2">7/10</div></div>
 						<div class="Text3">
-<Blue>Manufacture <i>CP<Super>+</Super></i> plate</Blue>
+<ol class="part1">
-						</div>
+	<li>DNA (insert & vector)</li>
+	<li>Ligation high ver.2</li>
+</ol>
+						</div>
+					<div class="Text2">
+<Green>Equipment :</Green>
+					</div>
 						<div class="Text3">
-<Blue>Manufacture <i>Amp<Super>+</Super></i> plate</Blue>
+<ol class="part1">
+	<li>Cooling dry bath incubator</li>
+</ol>
 						</div>
+					<div class="Text2">
+<Green>Procedure :</Green>
+					</div>
 						<div class="Text3">
-<Green>Suicide Part:</Green>
+<ol class="part2">
-						</div>
+	<li>
-						<div class="Text3_Sub">
+		Table
-Gel electrophoresis:
+		<table>
-						</div>
+			<tr>
-						<div class="Text3_SubSub">
+				<th>Components</th>
-[RBS] (BBa_B0034) ----- Result: Failed (Smeared band was too blurred)
+				<th>Volume (μL)</th>
-						</div>
+			</tr>
-						<div class="Text3_SubSub">
+			<tr>
-[C2] (BBa_C0053) ----- Result: Succeeded
+				<td>Insert</td>
-						</div>
+				<td>x</td>
-						<div class="Text3_Sub">
+			</tr>
-Gel extraction:
+			<tr>
-						</div>
+				<td>Vector</td>
-						<div class="Text3_SubSub">
+				<td>y</td>
-[C2]
+			</tr>
+			<tr>
+				<td>Ligation High</td>
+				<td>1</td>
+			</tr>
+			<tr class="alt">
+				<td>Total</td>
+				<td>7</td>
+			</tr>
+		</table>
+		&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Molar ratio: insert/vector = 3/1
+		<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; x + y = 6</br>
+	</li>
+	<li>Gently mix the solution by pipetting up and down</li>
+	<li>Incubate
+		<ol class="part3">
+			<li>at 16℃ for 2 hours, or</li>
+			<li>at 37℃ for 1 hour, or</li>
+			<li>at 4℃ overnight</li>
+		</ol>
+	</li>
+	<li>Proceed with bacterial transformation</li>
+</ol>
 						</div>
+				<div class="Text1" id="First6"><Red>Plasmid DNA Extraction Using Alkaline Lysis Method (<i>E. coli</i>)</Red></div>
-					<div class="Container2"><div class="Text2">7/11</div></div>
+					<div class="Text2">
+						<Green>Materials and Reagents :</Green>
+					</div>
 						<div class="Text3">
-<Green>Suicide Part:</Green>
+<ol class="part1">
-						</div>
+	<li><i>Escherichia coli</i> DH5α (with plasmid)</li>
-						<div class="Text3_Sub">
+	<li>LB broth</li>
-Extract the plasmid:
+	<li>Resuspension solution (MPI, with RNAase)</li>
-						</div>
+	<li>Lysis solution (MPII)</li>
-						<div class="Text3_SubSub">
+	<li>Neutralizing solution (MPIII)</li>
-[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)
+	<li>Isopropanol</li>
-						</div>
+	<li>70% ethanol</li>
+	<li>ddH<Sub>2</Sub>O</li>
+</ol>
+						</div>
+					<div class="Text2">
+						<Green>Equipment :</Green>
+					</div>
 						<div class="Text3">
-<Green>PYY part:</Green>
+<ol class="part1">
+	<li>Shaker</li>
+	<li>Centrifuge</li>
+	<li>Vortex mixer</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+					<div class="Text2">
-Transformation of:
+						<Green>Procedure :</Green>
-						</div>
+					</div>
-						<div class="Text3_SubSub">
-[promoter] (BBa_J23101)
-						</div>
-					<div class="Container2"><div class="Text2">7/12</div></div>
 						<div class="Text3">
-<Green>Suicide Part:</Green>
+<ol class="part2">
+        <li>Grow bacteria in LB broth with appropriate antibiotics at 37℃ overnight with shaking</li>
+	<li>Transfer 1.5mL culture to an eppendorf</li>
+	<li>Centrifuge at 4℃, 5000rpm for 5 minutes</li>
+	<li>Discard the supernatant</li>
+        <li>Add 150μL of resuspension solution (MPI, with RNAase) into each tube, pipet several times, and vortex to completely resuspend the cell pellet</li>
+        <li>Add 150μL of lysis solution (MPII), gently invert the tubes about 20 times, and then let the sample mixture stand at room temperature for 2 minutes</li>
+        <li>Add 150μL of neutralizing solution (MPIII) and mix by inverting the tubes about 20 times. Bacterial chromosomal DNA and proteins can be seen as white precipitates</li>
+        <li>Centrifuge at 4℃, 15000rpm for 15 minutes</li>
+        <li>Carefully transfer 400μL of the supernatant to a new eppendorf</li>
+        (Step 8&9 can be done twice)
+        <li>Add 400μL (same volume as the supernatant) isopropanol, and shake up the tubes as vigorously as one can</li>
+        <li>Centrifuge at 4℃, 15000rpm for 15 minutes</li>
+        <li>Discard the supernatant</li>
+        <li>Add 200μL of 70% ethanol to wash out the salts</li>
+        <li>Centrifuge at 4℃, 15000rpm for 5 minutes</li>
+        <li>Discard the supernatant and remove the remains as much as possible with pipetman</li>
+        <li>Air dry for about 30 minutes</li>
+        <li>Resuspend the DNA pellet with 20μL ddH<Sub>2</Sub>O</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+				<div class="Text1" id="First7"><Red>Transformation (<i>E. coli</i>)</Red></div>
-Extract the plasmid:
+					<div class="Text2">
-						</div>
+						<Green>Materials and Reagents :</Green>
-						<div class="Text3_SubSub">
+					</div>
-[pC1] (BBa_R0051)
-						</div>
-					<div class="Container2"><div class="Text2">7/13</div></div>
 						<div class="Text3">
-<Green>PYY part:</Green>
+<ol class="part1">
-						</div>
+	<li>Competent cell (<i>Escherichia coli</i> DH5α)</li>
-						<div class="Text3_Sub">
+	<li>LB plate (Amp+/CP+)</li>
-Extract the plasmid:
+</ol>
 						</div>
-						<div class="Text3_SubSub">
+<div class="Text2">
-[promoter] (BBa_J23101)
+<Green>Equipment :</Green>
-						</div>
+					</div>
-						<div class="Text3_Sub">
-Gel electrophoresis (two white colonies & two red colonies):
-						</div>
-						<div class="Text3_SubSub">
-[promoter] ------ Result: Failed (white colonies didn’t have band, red colonies had band but not the correct size, we speculated that was pollution)
-						</div>
-					<div class="Container2"><div class="Text2">7/20</div></div>
 						<div class="Text3">
-<Red>Discuss about total synthesis</Red>
+<ol class="part1">
+	<li>-80℃ refrigerator</li>
+	<li>Ice</li>
+	<li>37℃ incubator</li>
+	<li>Super optimal broth (SOB) (37℃)</li>
+	<li>Shaker</li>
+</ol>
 						</div>
+					<div class="Text2">
+						<Green>Procedure :</Green>
-					<div class="Container2"><div class="Text2">7/13</div></div>
+					</div>
 						<div class="Text3">
-<Green>PYY part:</Green>
+<ol class="part2">
-						</div>
+        <li>Take out the competent cells from -80℃ refrigerator</li>
-						<div class="Text3_Sub">
+        <li>Thaw the cells on ice for 5 minutes</li>
-Transformation of:
+        <li>Add 1μL plasmid DNA into the competent cells, and mix gently by pipetting</li>
-						</div>
+        <li>On ice for 10 minutes</li>
-						<div class="Text3_SubSub">
+        <li>Heat shock at 37℃ for 3 minutes</li>
-[terminator] (BBa_B0015)
+        <li>On ice for 2 minutes</li>
-						</div>
+        <li>Add 150μL 37℃ SOB into the mixture</li>
+        <li>Place the tube at 37℃ with shaking for 1 hour(Amp+)/4 hours(CP+) respectively</li>
+        <li>Spread the cells onto the LB plates (Amp+/ CP+)</li>
+        <li>Incubate the plates at 37℃ with shaking for 12-16 hours</li>
+</ol>
-					<div class="Container2"><div class="Text2">7/24</div></div>
-						<div class="Text3">
-<Red>Discuss our project with professor</Red>
 						</div>
+				<div class="Text1" id="First8"><Red>Protein Extraction</Red></div>
+					<div class="Text2">
+<Green>Materials and Reagents :</Green>
+					</div>
 						<div class="Text3">
-<Green>PYY part:</Green>
+<ol class="part1">
+	<li>plasmid/ BL21 competent cell</li>
+	<li>LB plate</li>
+	<li>LB broth</li>
+	<li>lysis buffer</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+					<div class="Text2">
-Extract the plasmid:
+<Green>Equipment :</Green>
-						</div>
+					</div>
-						<div class="Text3_SubSub">
-[terminator] (BBa_B0015)
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text3_SubSub">
-[terminator] ----- Result: Failed
-						</div>
-					<div class="Container2"><div class="Text2">7/25</div></div>
 						<div class="Text3">
-<Red>Discuss our problems with Instructor</Red>
+<ol class="part1">
+	<li>15/ 50ml conial tube</li>
+	<li>Shaking incubator</li>
+	<li>Erlenmeyer flask</li>
+	<li>Spectrophotometer</li>
+	<li>Centrifuge</li>
+	<li>Sonicator</li>
+	<li>Eppendorf</li>
+</ol>
 						</div>
+					<div class="Text2">
+<Green>Procedure :</Green>
-					<div class="Container2"><div class="Text2">7/26</div></div>
+					</div>
 						<div class="Text3">
-<Green>PYY part:</Green>
+<ol class="part2">
+	<li>Transform plamid into BL21 (see transformation)</li>
+	<li>Pick up a single colony and incubate into 2ml LB/CP medium, then agitate them in a shaking incubator at 37℃ overnight</li>
+	<li>Pour the culture into 100ml LB/CP medium, then agitate until it reach A<sub>600</sub> of 0.6</li>
+	<li>Pour the culture into 50ml conical tube, and centrifuge at 8000rpm for 10 min</li>
+	<li>Store the supernate and the pellets in the centrifuge tubes in a -80℃ freezer</li>
+	<li>Use 6ml lysis buffer(NP-10) to resuspend the cell pellet and transfer into a 15ml centrifuge tube</li>
+	<li>Lyse the cell with sonication</li>
+	<li>Centrifuge 15ml conial tube at 9000rpm for 10 min</li>
+	<li>Store the supernate as coarse extract</li>
+</ol>
 						</div>
-						<div class="Text3_SubSub">
+				<div class="Text1" id="First9"><Red>SDS-PAGE</Red></div>
-Cultured [terminator] again
+					<div class="Text2">
-						</div>
+<Green>Materials and Reagents :</Green>
+					</div>
-					<div class="Container2"><div class="Text2">7/27</div></div>
 						<div class="Text3">
-<Green>PYY part:</Green>
+<ol class="part1">
-						</div>
+	<li>Solution A (acrylamide/ bis-acrylamide (29:1) 30% solution)</li>
-						<div class="Text3_Sub">
+	<li>Solution B (Tris (Tris base, 1.5 M) 90.8 gm; TEMED 1.8 ml; pH = 8.8/ 500ml)</li>
-Extract the plasmid:
+	<li>Solution C (Tris (Tris base, 0.5 M) 6.0 gm; TEMED 0.4 ml; pH = 6.7/ 100ml)</li>
-						</div>
+	<li>10% SDS (1kb/100bp)</li>
-						<div class="Text3_SubSub">
+	<li>ddH<Sub>2</Sub>O</li>
-[terminator] (BBa_B0015)
+	<li>APS</li>
-						</div>
+	<li>Running buffer (1M Tris 12.5 ml; glysine 7.7g/ 500ml)</li>
-						<div class="Text3_Sub">
+	<li>Sample buffer(5X) (Tris base, 125mM X5; EDTA2•Na, 2mM X 5; SDS(2% X2); β-mercaptoethanol, 5% X5)</li>
-Gel electrophoresis:
+	<li>Protein marker</li>
-						</div>
+</ol>
-						<div class="Text3_SubSub">
+						</div>
-[terminator] ----- Result: Succeeded
+					<div class="Text2">
-						</div>
+<Green>Equipment :</Green>
+					</div>
-					<div class="Container2" id="First2"><div class="Text2">8/2</div></div>
 						<div class="Text3">
-<Green>Suicide Part:</Green>
+<ol class="part1">
+	<li>Gel Caster</li>
+	<li>Accessories for each gel</li>
+	<li>&nbsp;&nbsp;&nbsp;&nbsp;(1) One glass plate</li>
+	<li>&nbsp;&nbsp;&nbsp;&nbsp;(2) One aluminum notched plate</li>
+        <li>&nbsp;&nbsp;&nbsp;&nbsp;(3) Two 0.75-mm thick spacers</li>
+        <li>&nbsp;&nbsp;&nbsp;&nbsp;(4) One 0.75-mm thick 10-well comb</li>
+        <li>&nbsp;&nbsp;&nbsp;&nbsp;(5) One well-locating decal</li>
+	<li>Power supply</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+					<div class="Text2">
-Extract the plasmid:
+<Green>Procedure :</Green>
-						</div>
+					</div>
-						<div class="Text3_SubSub">
-[RFP](BBa_E1010)
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[RFP] cleave by EcoRI and SpeI
-						</div>
-					        <div class="Text3_SubSub">
-[Cl] cleave by XbaI and PstI
-						</div>
-					        <div class="Text3_SubSub">
-[pSB1A3] cleave by EcoRI and PstI
-						</div>
-					<div class="Container2"><div class="Text2">8/5</div></div>
 						<div class="Text3">
-<Blue>Passaged L.c.393</Blue>
+<ol class="part2">
+	<li>Assemble one glass plate and one alumimum plate with   two spacers for each gel module</li>
+	<li>Prepare the SDS gel solution (see the table below). Two 0.75mm mini gels need approximately 10ml separation gel solution and 5ml stacking solution. Add APS solution at the final step</li>
+        <li>Mix the separation gel solution, and add the solution into the gel module until reaching the designated level. Then add isopropanol on the top of the gel. It takes about 40 min to complete the polymerization step</li>
+        <li>Remove the isopropanol by using a small strip of filter paper. Fill up the stacking gel solution until reaching the top. Insert comb into the gel solution as soon as possible. It takes 10 min to complete the polymerization step</li>
+        <li>Remove the comb and set the gel in the cell buffer dam. Pour the running buffer into the inner chamber and keep pouring after overflow until the buffer surface reaches the required level in the outer chamber</li>
+        <li>Use a micropipette to clean up every single well with the running buffer</li>
+        <li>Mix protein samples with 1/5 volumes of 5X sample buffer. Heat the mixture at 100oC for 10 min. Let it cool down to room temperature</li>
+        <li>Load protein sample and the protein marker into the well</li>
+        <li>Run the electrophoresis at 70V for 20 min, and then 150V for 55 min</li>
+        <li>Remove the two spacer and lift the glass plate, and remove the stacking gel and the remaining gel is ready for CBR staining and Western blot</li>
+</ol>
 						</div>
 						<div class="Text3">
-<Blue>Manufacture MRS plate</Blue>
+		<table class="Table_Width">
+			<tr>
+				<th>(Unit: ml)</th>
+				<th>Separation gel</th>
+				<th></th>
+				<th></th>
+				<th></th>
+				<th></th>
+				<th></th>
+				<th>Stacking gel</th>
+			</tr>
+			<tr>
+				<td>Percentage</td>
+				<td>5%</td>
+				<td>7.5%</td>
+				<td>10%</td>
+				<td>12.5%</td>
+				<td>15%</td>
+				<td>20%</td>
+				<td>4%</td>
+			</tr>
+			<tr>
+				<td>Solution A</td>
+				<td>16.5</td>
+				<td>2.5</td>
+				<td>3.35</td>
+				<td>4.15</td>
+				<td>5</td>
+				<td>6.5</td>
+				<td>0.66</td>
+			</tr>
+			<tr>
+				<td>Solution B</td>
+				<td>2.5</td>
+				<td>2.5</td>
+				<td>2.5</td>
+				<td>2.5</td>
+				<td>2.5</td>
+				<td>2.5</td>
+				<td>-</td>
+			</tr>
+			<tr>
+				<td>Solution C</td>
+				<td>-</td>
+				<td>-</td>
+				<td>-</td>
+				<td>-</td>
+				<td>-</td>
+				<td>-</td>
+				<td>1.24</td>
+			</tr>
+			<tr>
+				<td>10% SDS</td>
+				<td>0.1</td>
+				<td>0.1</td>
+				<td>0.1</td>
+				<td>0.1</td>
+				<td>0.1</td>
+				<td>0.1</td>
+				<td>0.05</td>
+			</tr>
+			<tr>
+				<td>dH<sub>2</sub>O</td>
+				<td>5.7</td>
+				<td>4.85</td>
+				<td>4</td>
+				<td>3.2</td>
+				<td>2.35</td>
+				<td>0.85</td>
+				<td>2.95</td>
+			</tr>
+			<tr>
+				<td>APS(10%)</td>
+				<td>0.05</td>
+				<td>0.05</td>
+				<td>0.05</td>
+				<td>0.05</td>
+				<td>0.05</td>
+				<td>0.05</td>
+				<td>0.1</td>
+			</tr>
+			<tr class="alt">
+				<td>Total Volume</td>
+				<td>10</td>
+				<td>10</td>
+				<td>10</td>
+				<td>10</td>
+				<td>10</td>
+				<td>10</td>
+				<td>5</td>
+			</tr>
+		</table>
 						</div>
+				<div class="Text1" id="First10"><Red>Western Blot</Red></div>
+					<div class="Text2">
+						<Green>Materials and Reagents :</Green>
+					</div>
 						<div class="Text3">
-<Green>PYY part</Green>
+<ol class="part1">
-						</div>
+	<li>Transfer buffer ( Tris 25mM; glycine 0.192M)</li>
-						<div class="Text3_Sub">
+	<li>Methanol</li>
-Redissoluted DNA of total synthesis
+	<li>10 X TBST (1M Tris pH =8.0; Tween 20, 50ml; NaCl 43.5g)</li>
-						</div>
+</ol>
-						<div class="Text3_Sub">
+						</div>
-Restriction enzyme:
+					<div class="Text2">
-						</div>
+						<Green>Equipment :</Green>
-					        <div class="Text3_SubSub">
+					</div>
-[signalpeptide -PYY] cleave by EcoRI and PstI
-						</div>
-					        <div class="Text3_Sub">
-Ligation:
-						</div>
-					        <div class="Text3_SubSub">
-[signalpeptide-PYY]+ [pSB1C3]
-						</div>
-					        <div class="Text3_Sub">
-Transformation
-						</div>
-					<div class="Container2"><div class="Text2">8/6</div></div>
 						<div class="Text3">
-<Blue>Passaged L.c.393</Blue>
+<ol class="part1">
+	<li>Semi-dry trnasfer cell</li>
+	<li>PVDF membrane</li>
+	<li>Nitrocellulose paper</li>
+	<li>Square Petri dish</li>
+	<li>Rotary shaker</li>
+</ol>
 						</div>
+					<div class="Text2">
+						<Green>Procedure :</Green>
+					</div>
 						<div class="Text3">
-<Green>PYY part</Green>
+<ol class="part2">
+        <li>Equilibrate the nitrocellulose paper and SDS-PAGE gel by soaking them with transfer buffer</li>
+	<li>Rinse the PVDF membrane with 100% methanol for a few second and equilibrate it with transfer buffer</li>
+	<li>Place the elements in the following order onto the semi-dry transfer cell from bottom to top: nitrocellulose paper, PVDF membrane, SDS-PAGE gel, nitrocellulose paper</li>
+	<li>Set up the current at 0.09A and run for 75 min</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+				<div class="Text1" id="First11"><Red>Immunostaining</Red></div>
-Restriction enzyme:
+					<div class="Text2">
-						</div>
+<Green>Materials and Reagents :</Green>
-						<div class="Text3_SubSub">
+					</div>
-[PYY] cleave by EcoRI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by EcoRI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[Y2R] cleave by KpnI and XhoI
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
-						<div class="Text3_SubSub">
-[PYY]+ [pSB1C3]
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY]+ [pSB1C3]
-						</div>
-						<div class="Text3_SubSub">
-[Y2R]+[pcDNA]
-						</div>
-						<div class="Text3_Sub">
-Transformation
-						</div>
-					<div class="Container2"><div class="Text2">8/7</div></div>
 						<div class="Text3">
-<Green>PYY part</Green>
+<ol class="part1">
-						</div>
+	<li>3% milk</li>
-						<div class="Text3_Sub">
+	<li>Antibody solution</li>
-Gel electrophoresis:
+	<li>Secondary antibody (anti-mouse HRP)</li>
-						</div>
+	<li>TBST</li>
-						<div class="Text_underline">
+	<li>Western HRP substrate</li>
-Result:
+</ol>
 						</div>
-						<div class="Text3_SubSub">
+					<div class="Text2">
-signalpeptide-PYY] ----- Failed (Smeared band was too blurred)
+<Green>Equipment :</Green>
-						</div>
+					</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Failed (Smeared band was too blurred)
-						</div>
-						<div class="Text3_Sub">
-Transform again
-						</div>
-					<div class="Container2"><div class="Text2">8/10</div></div>
 						<div class="Text3">
-<Blue>Manufacture CP<sup>+</sup> plate</Blue>
+<ol class="part1">
+	<li>Square petri dish</li>
+	<li>Rotary shaker</li>
+	<li>UVP Biospectrum</li>
+</ol>
 						</div>
+					<div class="Text2">
+<Green>Procedure :</Green>
+					</div>
 						<div class="Text3">
-<Green>Suicide part</Green>
+<ol class="part2">
-						</div>
+	<li>Put the membrane in the square petri dish block the membrane with the 3% milk/TBST at room temperature for 1hr</li>
-						<div class="Text3_Sub">
+	<li>Discard the milk and replace it with antibody solution(mouse His-probe Antibody) at 4℃ overnight</li>
-Gel electrophoresis:
+        <li>Recycle the antibody solution. Wash the membrane with TBST three times, 5 min each</li>
-						</div>
+        <li>Soak membrane into anti-mouse HRP solution for 1 hr</li>
-						<div class="Text_underline">
+        <li>Discard the anti-mouse HRP solution and wash it with TBST three times, 5 min each</li>
-Result:
+        <li>Add HRP substrate on the membrane for 1 min</li>
-						</div>
+        <li>Soak the membrane into ddH<sub>2</sub>O to remove the substrate</li>
-						<div class="Text3_SubSub">
+        <li>Observe the result with UVP Biospectrum</li>
-[RFP+Terminator] ----- Failed (Confirmed it was pollution)
+</ol>
-						</div>
-						<div class="Text3_SubSub">
-[Cl+Terminator] ----- Failed
-						</div>
-						<div class="Text3_Sub">
-Clean up:
-						</div>
-						<div class="Text3_Sub">
-[RFP+Terminator]
 						</div>
+				<div class="Text1" id="First12"><Red>Thawing Caco-2 cells</Red></div>
+					<div class="Text2">
+<Green>Materials and Reagents :</Green>
+					</div>
 						<div class="Text3">
-<Green>PYY part</Green>
+<ol class="part1">
-						</div>
+	<li>1 ml frozen Caco-2 cells in cryovial</li>
-						<div class="Text3_Sub">
+	<li>Cell culture medium (MEM (minimum essential medium) (GIBCOTM #41500-034)/2.2g NaHCO3/ 0.11 g sodium pyruvate/20% FBS/1% PSA) </li>
-Clean up:
+	<li>70% ethanol</li>
-						</div>
+	<li>Trypan blue solution</li>
-						<div class="Text3_SubSub">
+</ol>
-[signalpeptide-PYY]+ [pSB1A3]
+						</div>
-						</div>
+					<div class="Text2">
-						<div class="Text3_SubSub">
+<Green>Equipment :</Green>
-[PYY]+ [pSB1A3]
+					</div>
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY]+ [pSB1A3]
-						</div>
-						<div class="Text3_SubSub">
-[Y2R]+[pcDNA]
-						</div>
-						<div class="Text3_Sub">
-Transformation
-						</div>
-					<div class="Container2"><div class="Text2">8/11</div></div>
 						<div class="Text3">
-<Green>Suicide part</Green>
+<ol class="part1">
-						</div>
+	<li>Laminar flow hood</li>
-						<div class="Text3_Sub">
+	<li>Auto pipette</li>
-Clean up:
+	<li>Centrifuge</li>
-						</div>
+	<li>15 ml centrifuge tubes</li>
-						<div class="Text3_SubSub">
+	<li>10 cm dish</li>
-[Cl+Terminator]
+	<li>Hemocytometer</li>
-						</div>
+	<li>Optical microscope</li>
-						<div class="Text3_Sub">
+	<li>Suction machine</li>
-Gel electrophoresis
+</ol>
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[RFP+Terminator]----- Failed (Smeared band was too blurred)
-						</div>
-						<div class="Text3_SubSub">
-[Cl+Terminator] ----- Failed (Smeared band was too blurred)
 						</div>
+					<div class="Text2">
+<Green>Procedure :</Green>
+					</div>
 						<div class="Text3">
-<Green>PYY part</Green>
+<ol class="part2">
+	<li>Pre-warm medium in 37˚C water bath</li>
+	<li>Open a laminar flow hood</li>
+        <li>Clean materials, reagents and equipments before moving them into the hood</li>
+        <li>Thawing Caco-2 cells in 37˚C water bath and transfer the cells into a 15ml centrifuge tube</li>
+        <li>Add 9 ml cell culture medium very slowly</li>
+        <li>Centrifuge at 1000 rpm for 5 min</li>
+        <li>Decant the supernate and resuspend cells with 10 ml cell culture medium</li>
+        <li>Mix 10 ul cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer</li>
+        <li>Add all cell suspension to a 10 cm dish</li>
+        <li>Write date, cell line and passage number</li>
+        <li>Put the dish into incubator</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+				<div class="Text1" id="First13"><Red>Freezing Caco-2 cells</Red></div>
-Extract the plasmid:
+					<div class="Text2">
-						</div>
+<Green>Materials and Reagents :</Green>
-						<div class="Text3_SubSub">
+					</div>
-[TAT-PYY], [Y2R]
-						</div>
-					<div class="Container2"><div class="Text2">8/12</div></div>
 						<div class="Text3">
-<Green>Suicide part</Green>
+<ol class="part1">
-						</div>
+	<li>Caco-2 cell in 10 cm dish</li>
-						<div class="Text3_Sub">
+	<li>Cell culture medium (MEM (minimum essential medium) (GIBCOTM #10370-021)/10% FBS/ 2 mM glutamine</li>
-Redissoluted DNA of total synthesis
+	<li>1X PBS (phosphate buffer saline)</li>
-						</div>
+	<li>0.5% trypsin</li>
-						<div class="Text3_Sub">
+	<li>Trypan blue solution</li>
-Restriction enzyme:
+	<li>70% ethanol</li>
-						</div>
+	<li>Cell freezing media (50% FBS, 40% cell culture medium, 10% DMSO)</li>
-						<div class="Text3_SubSub">
+</ol>
-[Lac-RBS] cleave by EcoRI and PstI
+						</div>
-						</div>
+					<div class="Text2">
-						<div class="Text3_SubSub">
+<Green>Equipment :</Green>
-[NucA-Terminator] cleave by EcoRI and PstI
+					</div>
-						</div>
-						<div class="Text3_SubSub">
-[NIsI] cleave by EcoRI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[LcSPddhol] cleave by EcoRI and PstI
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
-						<div class="Text3_SubSub">
-[Lac-RBS]+ [pSB1C3]
-						</div>
-						<div class="Text3_SubSub">
-[NucA-Terminator]+ [pSB1C3]
-						</div>
-						<div class="Text3_SubSub">
-[NIsI]+ [pSB1C3]
-						</div>
-						<div class="Text3_SubSub">
-[LcSPddhol]+ [pSB1C3]
-						</div>
-						<div class="Text3_Sub">
-Transformation
-						</div>
 						<div class="Text3">
-<Green>PYY part</Green>
+<ol class="part1">
+	<li>Laminar flow hood</li>
+	<li>Auto pipette</li>
+	<li>Centrifuge</li>
+	<li>15 ml centrifuge tubes</li>
+	<li>10 cm dish</li>
+	<li>Hemocytometer</li>
+	<li>Optical microscope</li>
+	<li>Suction machine</li>
+	<li>Cryovials</li>
+	<li>Freezing apparatus</li>
+	<li>-80˚C refrigerator</li>
+	<li>Liquid nitrogen tank</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+					<div class="Text2">
-Gel electrophoresis:
+<Green>Procedure :</Green>
-						</div>
+					</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY]----- Failed(Confirmed it was pollution)
-						</div>
-						<div class="Text3_SubSub">
-[Cll] ----- Succeeded
-						</div>
-					<div class="Container2"><div class="Text2">8/13</div></div>
 						<div class="Text3">
-<Green>Suicide part</Green>
+<ol class="part2">
-						</div>
+	<li>Pre-warm medium in 37˚C water bath</li>
-						<div class="Text3_Sub">
+	<li>Open a laminar flow hood</li>
-Extract the plasmid:
+        <li>Clean materials, reagents and equipments before moving them into the hood</li>
-						</div>
+        <li>Take a 10 cm dish with Caco-2 cell</li>
-						<div class="Text3_SubSub">
+        <li>Discard the medium with a suction machine</li>
-[Lac-RBS]+ [pSB1C3]
+        <li>Wash the cells with 5 ml PBS</li>
-						</div>
+        <li>Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes</li>
-						<div class="Text3_SubSub">
+        <li>Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface</li>
-[NucA-Terminator]+ [pSB1C3]
+        <li>Transfer the cell suspension to a 15 ml centrifuge tube</li>
-						</div>
+        <li>Mix 10 μl cell suspension and 10 ul trypan blue solution and count total cell number with a hemocytometer</li>
-						<div class="Text3_SubSub">
+        <li>Add about 1x10<sup>6</sup> cells per cryovial, and add cell freezing media</li>
-[NIsI]+ [pSB1C3]
+        <li>Freeze the cryovials in a controlled rate freezing apparatus, decreasing the temperature approximately 1°C per minute in a -80°C refrigerator for more than 3 hour, then move them into liquid nitrogen tank</li>
-						</div>
+</ol>
-						<div class="Text3_Sub">
-Clean up:
-						</div>
-						<div class="Text3_SubSub">
-[Cl], [terminator], [RFP]
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
-						<div class="Text3_SubSub">
-[Cl]+ [terminator]
-						</div>
-						<div class="Text3_SubSub">
-[RFP]+ [terminator]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[Cl]+ [terminator] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[RFP]+ [terminator] ----- Succeeded
 						</div>
+				<div class="Text1" id="First14"><Red>Caco2 Cell passage</Red></div>
+					<div class="Text2">
+<Green>Materials and Reagents :</Green>
+					</div>
 						<div class="Text3">
-<Green>PYY part</Green>
+<ol class="part1">
-						</div>
+	<li>Caco-2 cell in 10 cm dish</li>
-						<div class="Text3_Sub">
+	<li>Cell culture medium (MEM (minimum essential medium) (GIBCOTM #10370-021)/10% FBS/ 2 mM glutamine</li>
-Extract the plasmid:
+	<li>1X PBS (phosphate buffer saline)</li>
-						</div>
+	<li>0.5% trypsin</li>
-						<div class="Text3_SubSub">
+	<li>Trypan blue solution</li>
-[LcSPddhol]+ [pSB1C3]
+	<li>70% ethanol</li>
-						</div>
+</ol>
-						<div class="Text3_Sub">
+						</div>
-Clean up:
+					<div class="Text2">
-						</div>
+<Green>Equipment :</Green>
-						<div class="Text3_SubSub">
+					</div>
-[TAT-PYY]
-						</div>
-					<div class="Container2"><div class="Text2">8/14</div></div>
 						<div class="Text3">
-<Blue>DNA sequencing:</Blue>
+<ol class="part1">
-						</div>
+	<li>Laminar flow hood</li>
-						<div class="Text3_Sub">
+	<li>Auto pipette</li>
-[SP-PYY], [PYY], [Y2R], [Lac-RBS], [NucA-Terminator], [NIsI], [LcSPddhol]
+	<li>Centrifuge</li>
+	<li>15 ml centrifuge tubes</li>
+	<li>10 cm dish</li>
+	<li>Hemocytometer</li>
+	<li>Optical microscope</li>
+	<li>Suction machine</li>
+</ol>
 						</div>
+					<div class="Text2">
+<Green>Procedure :</Green>
+					</div>
 						<div class="Text3">
-<Green>Suicide part</Green>
+<ol class="part2">
+	<li>Pre-warm medium in 37˚C water bath</li>
+	<li>Open a laminar flow hood</li>
+        <li>Clean materials, reagents and equipments before moving them into the hood</li>
+        <li>Take a 10 cm dish with Caco-2 cell</li>
+        <li>Discard the medium with a suction machine</li>
+        <li>Wash the cells with 5 ml PBS</li>
+        <li>Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes</li>
+        <li>Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface</li>
+        <li>Transfer the cell suspension to a 15 ml centrifuge tube</li>
+        <li>Mix 10 ul cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer</li>
+        <li>Transfer appropriate amounts of cell suspension to new dishes (1/4 X)</li>
+        <li>Write date, cell line and passage number</li>
+        <li>Put the dish into incubator</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+				<div class="Text1" id="First15"><Red>Seeding in transwell</Red></div>
-Ligation:
+					<div class="Text2">
-						</div>
+<Green>Materials and Reagents :</Green>
-						<div class="Text3_SubSub">
+					</div>
-[Cl]+ [terminator]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[Lac-RBS] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[NucA-Terminator] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[NIsI] ----- Succeeded
-						</div>
-						<div class="Text3_Sub">
-[LcSPddhol] ----- Succeeded
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[RFP-terminator]
-						</div>
-					<div class="Container2"><div class="Text2">8/15</div></div>
 						<div class="Text3">
-<Blue>Passaged L.c.393</Blue>
+<ol class="part1">
-						</div>
+	<li>Caco-2 cell in 10 cm dish</li>
+	<li>Cell culture medium (MEM (minimum essential medium) (GIBCOTM #41500-034)/2.2 g NaHCO3/ 0.11 g sodium pyruvate/20% FBS/1% PSA</li>
+	<li>1X PBS (phosphate buffer saline)</li>
+	<li>0.5% trypsin</li>
+	<li>Trypan blue solution</li>
+	<li>70% ethanol</li>
+</ol>
+						</div>
+					<div class="Text2">
+<Green>Equipment :</Green>
+					</div>
 						<div class="Text3">
-<Green>Suicide part</Green>
+<ol class="part1">
-						</div>
+	<li>Laminar flow hood</li>
-						<div class="Text3_Sub">
+	<li>Auto pipette</li>
-Extract the plasmid:
+	<li>Centrifuge</li>
-						</div>
+	<li>15 ml centrifuge tubes</li>
-						<div class="Text3_SubSub">
+	<li>10 cm dish</li>
-[Cl-terminator]
+	<li>Hemocytometer</li>
-						</div>
+	<li>Optical microscope</li>
-						<div class="Text3_Sub">
+	<li>Suction machine</li>
-Gel electrophoresis:
+	<li>6-well plate</li>
-						</div>
+	<li>Millicell plate insert (PIHP03050)</li>
-						<div class="Text_underline">
+</ol>
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[RFP-terminator] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[Cl-terminator] ----- Succeeded
 						</div>
+					<div class="Text2">
+<Green>Procedure :</Green>
+					</div>
 						<div class="Text3">
-<Green>PYY part</Green>
+<ol class="part2">
+	<li>Open 6-well plate, add 1.5 mL medium to each large well (6 wells) which are going to be used</li>
+	<li>Add 2 ml PBS/well to other wells</li>
+        <li>Put transwell in larger well</li>
+        <li>Take a 10 cm dish with Caco-2 cell</li>
+        <li>Discard the medium with a suction machine</li>
+        <li>Wash the cells with 5 ml PBS</li>
+        <li>Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes</li>
+        <li>Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface</li>
+        <li>Transfer the cell suspension to a 15 ml centrifuge tube</li>
+        <li>Mix 10 ul cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer</li>
+        <li>Cell count: 70/ 2 * orginal volume *100 (*10<sup>4</sup>)= 35 X 10<sup>4</sup> cell/mL (Seeding 42 x 10<sup>4</sup> cell/well)</li>
+        <li>Write date, cell line and passage number</li>
+        <li>Put the dish into incubator</li>
+</ol>
 						</div>
-						<div class="Text3_Sub">
+				<div class="Text1" id="First16"><Red>Measure TEER</Red></div>
-Extract the plasmid:
+					<div class="Text2">
-						</div>
+<Green>Materials and Reagents :</Green>
-						<div class="Text3_SubSub">
+					</div>
-[TAT-PYY]
-						</div>
-						<div class="Text3_Sub">
-Transformation of [TAT-PYY]
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by EcoRI and PstI
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
-						</div>
-					<div class="Container2"><div class="Text2">8/16</div></div>
 						<div class="Text3">
-<Blue>Passaged L.c.393</Blue>
+<ol class="part1">
-						</div>
+	<li>Caco-2 cells seeded in transwell</li>
+	<li>70% ethanol</li>
+</ol>
+						</div>
+					<div class="Text2">
+<Green>Equipment :</Green>
+					</div>
 						<div class="Text3">
-<Blue>Manufacture LB broth</Blue>
+<ol class="part1">
+	<li>Laminar flow hood</li>
+	<li>Millicell ERS</li>
+</ol>
 						</div>
+					<div class="Text2">
+<Green>Procedure :</Green>
+					</div>
 						<div class="Text3">
-<Blue>Manufacture MRS broth</Blue>
+<ol class="part2">
-						</div>
+	<li>Prepare laminar hood for experiment</li>
-						<div class="Text3">
+	<li>To sterilize electrode of Millicell ERS, soak electrode in 70% alcohol for 15 min, and air dry for 15 sec</li>
-<Green>Suicide part</Green>
+        <li>Measure TEER value of each well (n=3)</li>
- 						</div>
+</ol>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[Cl-Terminator] cleave by EcoRI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[RFP-Terminator] cleave by EcoRI and PstI
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Transformation of [SP-PYY], [PYY], [TAT-PYY]
 						</div>
-					<div class="Container2"><div class="Text2">8/17</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
-						<div class="Text3_SubSub">
-[plac-RFP-T], [plac-Cl-T]
-						</div>
-						<div class="Text3_Sub">
-Transformation of [plac-RFP-T], [plac-Cl-T]
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Culture [SP-PYY], [PYY], [TAT-PYY] in BL21 Competent
-						</div>
-						<div class="Text3_Sub">
-<i>E. coli</i> and Rosetta Competent <i>E. coli</i>
-						</div>
-					<div class="Container2"><div class="Text2">8/18</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Culture [plac-RFP], [plac-Cl], [NucA] in LB broth
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[plac-RFP], [plac-Cl]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[plac-RFP] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[plac-Cl] ----- Succeeded
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY]):
-						</div>
-						<div class="Text3_SubSub">
-Sonication of Competent <i>E. coli</i>
-						</div>
-					<div class="Container2"><div class="Text2">8/19</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[NucA]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[NucA] ----- Succeeded
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY]):
-						</div>
-						<div class="Text3_SubSub">
-SDS-page
-						</div>
-						<div class="Text3_SubSub">
-Transfer
-						</div>
-						<div class="Text3_SubSub">
-Blocking
-						</div>
-						<div class="Text3_SubSub">
-Primary antibody
-						</div>
-					<div class="Container2"><div class="Text2">8/20</div></div>
-						<div class="Text3">
-<Blue>Passaged Caco2 cell</Blue>
-						</div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Gel Purification:
-						</div>
-						<div class="Text3_SubSub">
-[plac-Cl-T], [pCl-NucA-T]
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
-						<div class="Text3_SubSub">
-[plac-Cl-T], [pCl-NucA-T]
-						</div>
-						<div class="Text3_Sub">
-Transformation of [plac-Cl-T], [pCl-NucA-T]
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY]):
-						</div>
-						<div class="Text3_SubSub">
-Secondary antibody
-						</div>
-						<div class="Text3_SubSub">
-Chemiluminescent detection
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY]):
-						</div>
-						<div class="Text3_SubSub">
-SDS-page
-						</div>
-						<div class="Text3_SubSub">
-Transfer
-						</div>
-						<div class="Text3_SubSub">
-Blocking
-						</div>
-						<div class="Text3_SubSub">
-Primary antibody
-						</div>
-					<div class="Container2"><div class="Text2">8/21</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Culture [[plac-Cl-T], [pCl-NucA-T] in LB broth
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[plac-Cl-T], [pCl-NucA-T]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[plac-Cl-T] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[pCl-NucA-T] ----- Failed (band was too blurred)
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY]):
-						</div>
-						<div class="Text3_SubSub">
-Secondary antibody
-						</div>
-						<div class="Text3_SubSub">
-Chemiluminescent detection
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Failed (band was too blurred)
-						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_Sub">
-Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again. (use [RFP] as negative control)
-						</div>
-						<div class="Text3_Sub">
-Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21
-						</div>
-						<div class="Text3_Sub">
-Competent <i>E. coli</i> and Rosetta Competent <i>E. coli</i>
-						</div>
-					<div class="Container2"><div class="Text2">8/22</div></div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY] , [RFP]):
-						</div>
-						<div class="Text3_SubSub">
-Sonication of Competent <i>E. coli</i>
-						</div>
-					<div class="Container2"><div class="Text2">8/23</div></div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY] , [RFP]):
-						</div>
-						<div class="Text3_SubSub">
-SDS-page
-						</div>
-						<div class="Text3_SubSub">
-Transfer
-						</div>
-						<div class="Text3_SubSub">
-Blocking
-						</div>
-						<div class="Text3_SubSub">
-Primary antibody
-						</div>
-					<div class="Container2"><div class="Text2">8/24</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[NucA] cleave by SpeI and PstI
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY]):
-						</div>
-						<div class="Text3_SubSub">
-Secondary antibody
-						</div>
-						<div class="Text3_SubSub">
-Chemiluminescent detection
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Failed (band was not specificity)
-						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Failed (band was not specificity)
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Failed (band was not specificity)
-						</div>
-						<div class="Text3_Sub">
-Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP]in mini
-						</div>
-					<div class="Container2"><div class="Text2">8/25</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[plac-C1-T-backbone] cleave by EcoRI
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[plac-C1-T] ----- Failed
-						</div>
-						<div class="Text3_Sub">
-Redissoluted [pJ23102]
-						</div>
-						<div class="Text3_Sub">
-Transformation of [pCl], [pCll], [RBS], [pJ23102]
-						</div>
-						<div class="Text3_Sub">
-Culture [pCl], [pCll], [RBS] in LB broth
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in midi
-						</div>
-						<div class="Text3_Sub">
-Centrifugal and took the supernatant
-						</div>
-						<div class="Text3_Sub">
-Bradford protein assay
-						</div>
-					<div class="Container2"><div class="Text2">8/26</div></div>
- 						<div class="Text3">
-<Blue>Passaged Caco2 cell</Blue>
-						</div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
- 						<div class="Text3_SubSub">
-[plac-Cl-T]
-						</div>
-						<div class="Text3_Sub">
-Transformation of [plac-Cl-T]
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
- 						<div class="Text3_SubSub">
-[plac], [pCl], [pCll], [RBS]
-						</div>
-						 <div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
- 						</div>
- 						<div class="Text3_SubSub">
-SDS-page
-						</div>
-						 <div class="Text3_SubSub">
-Transfer
-						</div>
-						<div class="Text3_SubSub">
-Blocking
- 						</div>
-						<div class="Text3_SubSub">
-Primary antibody
-						</div>
-					<div class="Container2"><div class="Text2">8/27</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Transformation of [RBS-Cl-T], [RBS-RFP-T]
-						</div>
-						<div class="Text3_Sub">
-Culture [plac-Cl-T],[RBS-Cl-T],[RBS-RFP-T],[pJ23102] in LB broth
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[plac-Cl-T], [pJ23102]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[plac-Cl-T] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[pJ23102] ----- Succeeded
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[pCl-T] cleave by SpeI and PstI
-						</div>
-						<div class="Text3_Sub">
-Gel Purification:
-						</div>
-						<div class="Text3_SubSub">
-[pCll]
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
-						</div>
-						<div class="Text3_SubSub">
-Secondary antibody
-						</div>
-						<div class="Text3_SubSub">
-Chemiluminescent detection
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Failed (no band displayed)
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Failed (no band displayed)
- 						</div>
- 						<div class="Text3_Sub">
-Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again
- 						</div>
- 						<div class="Text3_Sub">
-Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21
-						</div>
- 						<div class="Text3_Sub">
-Competent <i>E. coli</i>
-						</div>
-					 <div class="Container2"><div class="Text2">8/28</div></div>
- 						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
- 						<div class="Text3_SubSub">
-[pCIt]+[NucA]
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
- 						<div class="Text3_SubSub">
-[Cl-T], [RFP-T]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
- 						 <div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[Cl-T] ----- Failed (band size was wrong)
-						</div>
-  						<div class="Text3_SubSub">
-[RFP-T] ----- Failed (band size was wrong)
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
- 						</div>
-  						<div class="Text3_SubSub">
-[plac-T] cleave by SpeI and PstI
-						</div>
-  						<div class="Text3_SubSub">
-[pJ23102] cleave by SpeI and PstI
-						</div>
-						<div class="Text3_Sub">
-Gel Purification:
-						</div>
- 						<div class="Text3_SubSub">
-[pCI-T]
-						</div>
-						 <div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
- 						</div>
- 						<div class="Text3_SubSub">
-SDS-page
-						</div>
-						<div class="Text3_SubSub">
-Transfer
-						</div>
-						<div class="Text3_SubSub">
-Blocking
- 						</div>
-						<div class="Text3_SubSub">
-Primary antibody
-						</div>
-					<div class="Container2"><div class="Text2">8/29</div></div>
-						<div class="Text3">
-<Blue>Passaged Caco2 cell</Blue>
-						</div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
- 						<div class="Text3_SubSub">
-[RBS-Cl-T], [RBS-RFP-T]
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
- 						</div>
-						<div class="Text3_SubSub">
-Secondary antibody
-						</div>
-						<div class="Text3_SubSub">
-Chemiluminescent detection
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_Sub">
-Histag column([SP-PYY], [PYY], [TAT-PYY] , [RFP])
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
- 						</div>
- 						<div class="Text3_SubSub">
-SDS-page
-						</div>
-						<div class="Text3_SubSub">
-Transfer
-						</div>
-						<div class="Text3_SubSub">
-Blocking
- 						</div>
-						<div class="Text3_SubSub">
-Primary antibody
-						</div>
-					<div class="Container2"><div class="Text2">8/30</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
- 						<div class="Text3_SubSub">
-[RBS], [pCl]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
- 						<div class="Text3_SubSub">
-[RBS] ----- Succeeded
-						</div>
- 						<div class="Text3_SubSub">
-[pCl] ----- Failed
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
- 						</div>
-						<div class="Text3_SubSub">
-Secondary antibody
-						</div>
-						<div class="Text3_SubSub">
-Chemiluminescent detection
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Failed (no band displayed)
-						</div>
-					<div class="Container2"><div class="Text2">8/31</div></div>
-						<div class="Text3">
-<Blue>Passaged hek293 cell</Blue>
-						</div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Gel Purification:
-						</div>
- 						<div class="Text3_SubSub">
-[RBS], [CI-T]
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
- 						</div>
- 						<div class="Text3_SubSub">
-SDS-page
-						</div>
-						<div class="Text3_SubSub">
-Transfer
-						</div>
-						<div class="Text3_SubSub">
-Blocking
- 						</div>
-						<div class="Text3_SubSub">
-Primary antibody
-						</div>
-					<div class="Container2" id="First3"><div class="Text2">9/1</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
- 						<div class="Text3_SubSub">
-[RBS] + [CI-T]
-						</div>
-						<div class="Text3_Sub">
-Culture [RBS- CI-T] in LB broth
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
- 						</div>
-						<div class="Text3_SubSub">
-Secondary antibody
-						</div>
-						<div class="Text3_SubSub">
-Chemiluminescent detection
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Failed (no band displayed)
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY] ----- Failed (no band displayed)
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Failed (no band displayed)
- 						</div>
- 						<div class="Text3_Sub">
-Transformation of [SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Culture [nisI] in MRS broth
-						</div>
-					<div class="Container2"><div class="Text2">9/2</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
- 						<div class="Text3_SubSub">
-[RBS- CI-T]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
- 						<div class="Text3_SubSub">
-[RBS- CI-T] ----- Succeeded
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] cleave by EcoRI
-						</div>
-						<div class="Text3_SubSub">
-[PYY] cleave by EcoRI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by EcoRI
-						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by EcoRI
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[pETDuet-1] ----- Succeeded
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Nisin Immunity Test
-						</div>
- 						<div class="Text3_Sub">
-Culture [nisI] in LB broth
-						</div>
-					<div class="Container2"><div class="Text2">9/3</div></div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Failed (small band didn’t display)
-						</div>
- 						<div class="Text3_SubSub">
-[PYY] ----- Failed (small band didn’t display)
-						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY] ----- Failed(small band didn’t display)
-						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] ----- Succeeded
-						</div>
-						<div class="Text3_Sub">
-Again -->
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[pETDuet-1] ----- Succeeded
- 						</div>
- 						<div class="Text3_Sub">
-℃ Ligation 4hr:
- 						</div>
-						<div class="Text3_SubSub">
-[SP-PYY]+ [pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY]+ [pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY]+ [pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
- 						<div class="Text3_SubSub">
-[nisI-pSB1C3]
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
- 						<div class="Text3_SubSub">
-[nisI-pSB1C3] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
- 						<div class="Text3_SubSub">
-[nisI-pSB1C3] ----- Succeeded
-						</div>
- 						<div class="Text3_Sub">
-Nisin Immunity Test
-						</div>
-					<div class="Container2"><div class="Text2">9/4</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
- 						<div class="Text3_SubSub">
-[pJ23102] + [RBS-CI-T]
-						</div>
-						<div class="Text3_SubSub">
-[plac] + [RBS-CI-T]
-						</div>
-						<div class="Text3_Sub">
-Transformation of [pJ23102-RBS-CI-T], [plac-RBS-CI-T]
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[pETDuet-1] ----- Succeeded
- 						</div>
- 						<div class="Text3_Sub">
-Gel Purification:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-℃ Ligation 4hr:
- 						</div>
-						<div class="Text3_SubSub">
-[SP-PYY]+ [pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY]+ [pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY]+ [pETDuet-1] ----- Failed
- 						</div>
-					<div class="Container2"><div class="Text2">9/5</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Culture[pJ23102-RBS-CI-T], [plac-RBS-CI-T] in LB broth
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[pETDuet-1] ----- Succeeded
- 						</div>
- 						<div class="Text3_Sub">
-Gel Purification:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-℃ Ligation 4hr:
- 						</div>
-						<div class="Text3_SubSub">
-[SP-PYY]+ [pETDuet-1]
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY]+ [pETDuet-1]
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY]+ [pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-Transformation of [SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-Measure the resistance of Caco2 cell
- 						</div>
-					<div class="Container2"><div class="Text2">9/6</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[placE-RBS-CI-T], [pJ23102-RBS-CI-T], [placE]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[placE-RBS-CI-T] ----- Failed
-						</div>
-						<div class="Text3_SubSub">
-[pJ23102-RBS-CI-T] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[placE] ----- Succeeded
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1],[PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_SubSub">
-[PYY-pETDuet-1] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet-1] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY-pETDuet-1] ----- Failed
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3_Sub">
-Extract the plasmid:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
- 						</div>
- 						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
- 						<div class="Text_underline">
-Result:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1]-----Failed(Ligation failed)
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY-pETDuet-1] ----- Failed(Ligation failed)
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet-1]-----Failed(Ligation failed)
- 						</div>
- 						<div class="Text3_Sub">
-Measure the resistance of Caco2 cell
- 						</div>
-					<div class="Container2"><div class="Text2">9/7</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[placE] cleave by SpeI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[RBS-CI-T] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_Sub">
-Transformation of [pCLP7]
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[pCLP7] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[nisi] cleave by XbaI and PstI
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
- 						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_SubSub">
-[PYY-pETDuet-1] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet-1] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Failed(small band didn’t display)
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY] ----- Failed(small band didn’t display)
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Failed(small band didn’t display)
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1]-----Failed(small band didn’t display)
- 						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1]-----Failed(Ligation failed)
- 						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] ----- Failed(Ligation failed)
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet-1]-----Failed(Ligation failed)
- 						</div>
- 						<div class="Text3_Sub">
-Measure the resistance of Caco2 cell
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
- 						</div>
- 						<div class="Text3_Sub">
-Construction of the plasmid:
- 						</div>
- 						<div class="Text3_SubSub">
-[nisI- pCLP7]
- 						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
- 						</div>
-						<div class="Text3_SubSub">
-[nisI- pCLP7] cleave by XbaI and PstI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
- 						<div class="Text_underline">
-Result:
- 						</div>
- 						<div class="Text3_SubSub">
-[nisI- pCLP7] ----- Succeeded
- 						</div>
- 						<div class="Text3_Sub">
-Clean up:
- 						</div>
- 						<div class="Text3_SubSub">
-[pCLP7]
- 						</div>
- 						<div class="Text3_Sub">
-Ligation:
- 						</div>
- 						<div class="Text3_SubSub">
-[nisI] + [pCLP7]
- 						</div>
- 						<div class="Text3_Sub">
-Transformation of [nisI- pCLP7]
- 						</div>
-					<div class="Container2"><div class="Text2">9/8</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[pCLP7] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[plac-RBS-RFP-T] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_Sub">
-Ligation:
-						</div>
-						<div class="Text3_SubSub">
-[pCLP7] + [plac-RBS-RFP-T]
-						</div>
-						<div class="Text3_Sub">
-Manufacture competent cell with [pJ23102-RBS-CI-T]
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
-						</div>
- 						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1]-----Succeeded
- 						</div>
- 						<div class="Text3_Sub">
-Gel Purification:
- 						</div>
-						<div class="Text3_SubSub">
-[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
- 						</div>
-						<div class="Text3_Sub">
-℃ Ligation overnight:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY]+ [pETDuet-1]
-						</div>
-						<div class="Text3_SubSub">
-[PYY]+ [pETDuet-1]
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY]+ [pETDuet-1]
-						</div>
-						<div class="Text3_Sub">
-Measure the resistance of Caco2 cell
- 						</div>
-						<div class="Text3_Sub">
-Transfect hek293 cell
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
- 						</div>
- 						<div class="Text3_Sub">
-Culture [nisI- pCLP7] in LB broth
- 						</div>
-					<div class="Container2"><div class="Text2">9/9</div></div>
-						<div class="Text3">
-<Blue>Passaged hek293 cell</Blue>
-						</div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[pCLP7], [place-RBS-CI-T]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[pCLP7] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[place-RBS-CI-T]-----Failed(concentration to low)
-						</div>
-						<div class="Text3_Sub">
-Transformation of [plac-RBS-RFP-T- pCIp]
-						</div>
-						<div class="Text3_Sub">
-Transformation of [plac-RBS-CI-T-NucA] in competent cell with [pJ23102-RBS-CI-T]
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Succeeded
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY] ----- Succeeded
-						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1]-----Succeeded
- 						</div>
- 						<div class="Text3_Sub">
-Measure the resistance of Caco2 cell
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
- 						</div>
- 						<div class="Text3_Sub">
-Extract the plasmid:
- 						</div>
- 						<div class="Text3_SubSub">
-[nisI- pCLP7]
- 						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
- 						</div>
- 						<div class="Text3_SubSub">
-[nisI- pCLP7] cleave by XbaI and PstI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
- 						<div class="Text_underline">
-Result:
- 						</div>
- 						<div class="Text3_SubSub">
-[nisI- pCLP7] ----- Failed
- 						</div>
-					<div class="Container2"><div class="Text2">9/10</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[plac-RBS-RFP-T- pCLP7]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[plac-RBS-RFP-T- pCLP7] ----- Failed
-						</div>
-						<div class="Text3_Sub">
-Manufacture L.casei competent cell
-						</div>
-						<div class="Text3_Sub">
-Electroporation of [pCIp] in L.casei competent cell
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
- 						<div class="Text3_Sub">
-Gel Purification:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
-						</div>
- 						<div class="Text3_Sub">
-℃ Ligation overnight:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY]+ [pETDuet-1]
- 						</div>
-						<div class="Text3_SubSub">
-[PYY]+ [pETDuet-1]
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY]+ [pETDuet-1]
- 						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1],[PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
-						</div>
-						<div class="Text3_Sub">
-Restriction enzyme:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY-pETDuet1] cleave by EcoRI and SpeI
-						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] cleave by EcoRI and SpeI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet1] cleave by EcoRI and SpeI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1] ----- Failed
- 						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] ----- Failed
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] ----- Succeeded
- 						</div>
- 						<div class="Text3_Sub">
-Gel Purification:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-Measure the resistance of Caco2 cell
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
- 						</div>
- 						<div class="Text3_Sub">
-Extract the plasmid:
- 						</div>
- 						<div class="Text3_SubSub">
-[nisI- pCLP7]
- 						</div>
- 						<div class="Text3_Sub">
-Nisin Immunity Test
- 						</div>
-					<div class="Container2"><div class="Text2">9/11</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[plac+RBS-RFP-T- pCLP7]
-						</div>
-						<div class="Text3_Sub">
-Gel electrophoresis:
-						</div>
-						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[plac+RBS-RFP-T- pCLP7] ----- Failed
-						</div>
-						<div class="Text3_Sub">
-Test electroporation of <i>L.casei</i>
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
- 						<div class="Text3_Sub">
-℃ Ligation overnight:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY]+ [pETDuet-1]
-						</div>
-						<div class="Text3_SubSub">
-[PYY]+ [pETDuet-1]
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY]+ [pETDuet-1]
- 						</div>
-						<div class="Text3_Sub">
-Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1] in XL1blue
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet1] cleave by XbaI and KpnI
- 						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] cleave by XbaI and KpnI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1] ----- Failed
- 						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] ----- Failed
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3_Sub">
-Extract the plasmid:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] cleave by EcoRI
-						</div>
-						<div class="Text3_SubSub">
-[PYY] cleave by EcoRI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by EcoRI
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by EcoRI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] ----- Succeeded
- 						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY] cleave by XbaI and PstI
-						</div>
-						<div class="Text3_SubSub">
-[PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] cleave by XbaI and PstI
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] cleave by XbaI and PstI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY] ----- Succeeded
- 						</div>
-						<div class="Text3_SubSub">
-[pETDuet-1] ----- Succeeded
- 						</div>
- 						<div class="Text3_Sub">
-Gel Purification:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-℃ Ligation overnight:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY]+ [pETDuet-1]
- 						</div>
- 						<div class="Text3_SubSub">
-[PYY]+ [pETDuet-1]
- 						</div>
- 						<div class="Text3_SubSub">
-[TAT-PYY]+ [pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-Measure the resistance of Caco2 cell
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
- 						</div>
- 						<div class="Text3_Sub">
-Nisin Immunity Test
- 						</div>
-					<div class="Container2"><div class="Text2">9/12</div></div>
-						<div class="Text3">
-<Green>Suicide part</Green>
-						</div>
-						<div class="Text3_Sub">
-Test absorbance(O.D. 600nm) of E.coli in Suicide circuit
-						</div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet1] cleave by XbaI and KpnI
- 						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] cleave by XbaI and KpnI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
- 						<div class="Text_underline">
-Result:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1] ----- Failed
- 						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] ----- Failed
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3_Sub">
-Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1] in XL1blue
- 						</div>
- 						<div class="Text3_Sub">
-Extract the plasmid:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
-						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
- 						</div>
- 						<div class="Text3_SubSub">
-[SP-PYY-pETDuet1] cleave by XbaI and KpnI
-						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] cleave by XbaI and KpnI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
- 						</div>
- 						<div class="Text3_Sub">
-Gel electrophoresis:
- 						</div>
-						<div class="Text_underline">
-Result:
- 						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1] ----- Failed
- 						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] ----- Failed
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet-1] ----- Failed
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
- 						</div>
- 						<div class="Text3_Sub">
-Nisin Immunity Test
- 						</div>
-					<div class="Container2"><div class="Text2">9/13</div></div>
-						<div class="Text3">
-<Green>PYY part</Green>
-						</div>
-						<div class="Text3_Sub">
-Extract the plasmid:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
- 						</div>
- 						<div class="Text3_Sub">
-Restriction enzyme:
-						</div>
-						<div class="Text3_SubSub">
-[SP-PYY-pETDuet1] cleave by XbaI and KpnI
- 						</div>
-						<div class="Text3_SubSub">
-[PYY-pETDuet-1] cleave by XbaI and KpnI
- 						</div>
-						<div class="Text3_SubSub">
-[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
- 						</div>
- 						<div class="Text3">
-<Green>Selection part</Green>
- 						</div>
- 						<div class="Text3_Sub">
-Nisin Immunity Test
- 						</div>
@@ Line 3,447: / Line 1,694: @@
 							Maintained by the iGEM team NTU-LIHPAO-Taiwan&nbsp;&nbsp;&nbsp;&nbsp;©2015 NTU-LIHPAO-Taiwan
 						</div>
 			</div>
 		</div>
@@ Line 3,462: / Line 1,706: @@
 			if (target.length) {
 				$('html,body').animate({
-					scrollTop: target.offset().top-55
+					scrollTop: target.offset().top-15
 				}, 700);
 				return false;
@@ Line 3,483: / Line 1,727: @@
 	});
-	$('.Text3 > a').click(function() {
+	$('.Text1 > a').click(function() {
 		console.log(this.hash);
 		if (location.pathname.replace(/^\//,'') == this.pathname.replace(/^\//,'') || location.hostname == this.hostname) {

Components	Volume (μL)
Insert	x
Vector	y
Ligation High	1
Total	7