Difference between revisions of "Team:CityU HK/Protocol"
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+ | /************* sidebar style **************/ | ||
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</div><!-- end .container --> | </div><!-- end .container --> | ||
</div><!-- end .header --> | </div><!-- end .header --> | ||
− | + | ||
+ | <!----------- sidebar ------------> | ||
+ | <div class="area"></div><nav class="main-menu"> | ||
+ | <ul> | ||
+ | <li> | ||
+ | <a href="#"> | ||
+ | <i class="fa"></i> | ||
+ | <span class="nav-text" id="first-item"> | ||
+ | PCR purification | ||
+ | </span> | ||
+ | </a> | ||
+ | |||
+ | </li> | ||
+ | <li class="has-subnav"> | ||
+ | <a href="#"> | ||
+ | <i class="fa"></i> | ||
+ | <span class="nav-text"> | ||
+ | Plasmid extraction | ||
+ | </span> | ||
+ | </a> | ||
+ | |||
+ | </li> | ||
+ | <li class="has-subnav"> | ||
+ | <a href="#"> | ||
+ | <i class="fa"></i> | ||
+ | <span class="nav-text"> | ||
+ | Gel purification | ||
+ | </span> | ||
+ | </a> | ||
+ | |||
+ | </li> | ||
+ | <li class="has-subnav"> | ||
+ | <a href="#"> | ||
+ | <i class="fa"></i> | ||
+ | <span class="nav-text"> | ||
+ | Protein purification | ||
+ | </span> | ||
+ | </a> | ||
+ | |||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#"> | ||
+ | <i class="fa fa-th-list fa-2x"></i> | ||
+ | <span class="nav-text"> | ||
+ | Quantitative RT-PCR | ||
+ | </span> | ||
+ | </a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#"> | ||
+ | <i class="fa"></i> | ||
+ | <span class="nav-text"> | ||
+ | Reverse transcription PCR | ||
+ | </span> | ||
+ | </a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#"> | ||
+ | <i class="fa"></i> | ||
+ | <span class="nav-text"> | ||
+ | RNA extraction | ||
+ | </span> | ||
+ | </a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#"> | ||
+ | <i class="fa"></i> | ||
+ | <span class="nav-text"> | ||
+ | ONPG assay | ||
+ | </span> | ||
+ | </a> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#"> | ||
+ | <i class="fa"></i> | ||
+ | <span class="nav-text"> | ||
+ | Colony PCR | ||
+ | </span> | ||
+ | </a> | ||
+ | </li> | ||
+ | </ul> | ||
+ | |||
+ | </nav> <!---------end of sidebar ---------> | ||
Revision as of 14:16, 15 September 2015
PCR Amplification
Protocol
Using TIANGEN 2× Pfu PCR MasterMix
PCR Reaction Mixture
|
Volume (μl) |
Master mix (μl) |
ddH2O |
9.5 |
39.9 |
Pfu PCR MasterMix
(2X) |
12.5 |
52.5 |
F-primer (10 mM) |
1 |
4.2 |
R-primer (10 mM) |
1 |
4.2 |
Total |
24 |
/ |
§ Add 1 μl of DNA template to 24 μl of master mix in each PCR tube.
Thermocycling Conditions
Step |
Temperature (oC) |
Time |
|
Initial denaturation |
94 |
3 minutes |
|
Denaturation |
94 |
30 seconds |
|
Annealing |
|
55 |
30 seconds |
Extension (This process is
repeated for 32 cycles) |
72 |
2 minutes* |
|
Final extension |
72 |
5 minutes |
|
Hold |
12 |
∞ |
|
*Depending on the size of amplicon (1,000 bp/minute)
PCR Amplification
Protocol
Using TIANGEN 2× Taq Plus PCR MasterMix
PCR Reaction Mixture
|
Volume (μl) |
Master mix (μl) |
ddH2O |
7.4 |
185 |
Taq Plus PCR MasterMix (2X) |
10 |
250 |
F-primer (10 mM) |
0.8 |
20 |
R-primer (10 mM) |
0.8 |
20 |
Total |
19 |
/ |
§ Add 1 μl of each DNA template to 19 μl of master mix in each PCR tube.
Thermocycling Conditions
Step |
Temperature (oC) |
Time |
|
Initial denaturation |
94 |
3 minutes |
|
Denaturation |
94 |
30 seconds |
|
Annealing |
|
55 |
30 seconds |
Extension (This process is
repeated for 32 cycles) |
72 |
1 minute* |
|
Final extension |
72 |
5 minutes |
|
Hold |
12 |
∞ |
|
*Depending on the size of amplicon (1,000 bp/minute)
</div>
</hr>
Purification of PCR
Products Protocol
Using TIANGEN® TIANquick Midi Purification Kit (DP204)
[1]
Add 500 μl
of Buffer BL to a CB2 spin column in a collection tube. Centrifuge for 1 minute
at 12,000 rpm (~13,400 x g). Discard the
flow-through.
[2]
Add 5 volumes of Buffer PB to 1
volume of nucleic acid solution in a 1.5 ml microcentrifuge
tube. Mix gently.
[3]
Transfer the mixture to the
spin column and incubate at room temperature for 2 minutes. Centrifuge for 1
minute at 12,000 rpm (~13,400 x g).
Discard the flow-through.
[4]
Add 600 μl
of Buffer PW to the spin column and centrifuge for 1 minute at 12,000 rpm (~13,400
x g). Discard the flow-through. Repeat.
[5]
Centrifuge the empty spin
column for 2 min.
[6]
Place the spin column in a
clean 1.5 ml microcentrifuge tube. Add 30 μl of Buffer EB or ddH2O (prewarmed at 60oC) to the center of the membrane. Incubate at room temperature for 2
minutes. Centrifuge at 12,000 rpm (~13,400 x g) for 2 minutes.
[7]
Repeat step 6 by using the
flow-through (DNA) in the 1.5 ml microcentrifuge
tube.
[8] Store the DNA at 4oC or –20oC.
</hr>
Plasmid Extraction Protocol
Using TIANGEN®
TIANprep Mini Plasmid Kit (DP103)
[1]
Add
500 μl of Buffer BL to spin column CP3 with
collection tube to activate the DNA-binding membrane. Centrifuge at 12,000 rpm
(~13,400 x g) for 1 minute. Discard the flow-through.
[2]
Harvest
a total of 3 ml of bacterial cells in a 1.5 ml microcentrifuge
tube by centrifuging 1.5 ml bacterial cells at 12,000 rpm (~13,400 x g)
for 1 minute and repeat.
[3]
Remove
the supernatant. Resuspend the cell pellet in 250 μl Buffer P1 until no cell crumbs can be seen.
[4]
Add
250 μl of Buffer P2 and mix thoroughly by inverting
the tube 6 to 8 times. (No vortexing)
[5]
Add
350 μl of Buffer P3 and immediately invert the tube 6
to 8 times. (No vortexing)
[6]
Centrifuge
the tube at 12,000 rpm (~13,400 x g) for 10 minutes.
[7]
Apply
the supernatant to the activated spin column and centrifuge for 1 minute at
12,000 rpm (~13,400 x g). Discard the flow-through.
[8]
Add
500 μl of Buffer PD to the spin column and centrifuge
for 1 minute at 12,000 rpm (~13,400 x g). Discard the flow-through.
[9]
Add
600 μl of Buffer PW to the spin column and centrifuge
for 1 minute at 12,000 rpm (~13,400 x g). Discard flow-through. Repeat.
[10]
Spin
the empty spin column for 2 minutes.
[11]
Place
the spin column in a clean 1.5 ml microcentrifuge
tube. Add 50 μl of Buffer EB or ddH2O (prewarmed at 60oC) to the center
of the membrane. Incubate at room temperature for 2 minutes. Centrifuge for 2
minutes at 12,000 rpm (~13,400 x g).
[12]
Repeat
step 11 by using the flow-through (DNA) in the 1.5 ml microcentrifuge
tube.
[13]
Store
the DNA at 4oC or –20oC.
</hr>
Purification of DNA from Gel Protocol
Using TIANGEN®
TIANgel Maxi DNA Purification Kit (DP210)
[1] Excise the desired DNA fragment from the agarose gel with a clean and sharp scalpel. Slice the gel piece into small pieces.
[2] Transfer the pieces into a microcentrifuge tube.
[3] Add 3 volumes of Buffer PN to 1 volume of gel pieces in a 1.5 ml microcentrifuge tube and mix well. Incubate the tubes in a 50oC waterbath until all gel pieces have completely dissolved.
[4] During the incubation, add 500 μl of Buffer BL to a CA3 spin column. Centrifuge at 12,000 rpm (~13,400 x g) for 1 minute and discard the flow-through.
[5] Apply the DNA mixture to the spin column (£ 800 μl) and incubate at room temperature for 2 minutes. Centrifuge 1 minute at 12,000 rpm (~13,400 x g). Discard the flow-through. Repeat to transfer the remaining DNA mixture if necessary.
[6] Add 600 μl of Buffer PW to the spin column and centrifuge for 1 minute at 12,000 rpm (~13,400 x g). Discard the flow-through. Repeat.
[7] Spin the empty spin column for 2 minutes.
[8] Place the spin column in a clean 1.5 ml microcentrifuge tube. Add 30 μl of Buffer EB or ddH2O (warmed at 60oC) to the center of membrane. Incubate at room temperature for 2 minutes. Centrifuge for 2 minutes at 12,000 rpm (~13,400 x g).
[9] Repeat step 8 by using the flow-through (DNA) in the 1.5 ml microcentrifuge tube.
[10] Store the DNA at 4oC or –20oC.
</hr>
</hr>
Colony PCR (analysis) Protocol
Using TaKaRa Ex Taq® DNA
Polymerase
Part I: Cell lysate
(template) preparation
[1]
Select well isolated colonies on
LB plates for PCR amplification. Prepare a PCR tube for each colony.
[2]
Pick colony with a sterile
pipette tip and resuspend colony in 5μl of sterile
ddH2O.
[3]
Heat PCR tubes in the thermocycler at 98oC for 10 minutes to lyse the
cells. And centrifuge the lysate at 14,000 rpm for 1 minute.
Part II: PCR
PCR Reaction Mixture
|
Volume of reaction mix (μl) for 1
reaction |
Volume of reaction mix (μl) for 12
reactions |
Ex Taq polymerase (5 units/μl) |
0.075 |
0.9 |
Taq PCR buffer
(10X) |
1.5 |
18 |
dNTPs (10 mM) |
1.2 |
14.4 |
VF2 primer (10 mM) |
0.3 |
3.6 |
VR primer (10 mM) |
0.3 |
3.6 |
ddH2O |
10.625 |
127.5 |
Total |
14 |
168 |
§ Mix 1 μl of DNA template (from step 3)
with 14 μl of master mix in a PCR tube.
Thermocycling Conditions
Step |
Temperature (oC) |
Time |
|
Initial denaturation |
98 |
2 minutes |
|
Denaturation |
98 |
30 seconds |
|
Annealing |
|
55 |
30 seconds |
Extension (This process is repeated for 32 cycles) |
72 |
2 minutes* |
|
Final extension |
72 |
1 minute |
|
Hold |
4 |
∞ |
|
*Depending on the size of amplicon (e.g. 2
minutes for 1 to 2 kb)
Colony PCR (analysis) Protocol
Using NEB Quick-Load® Taq
2X Master Mix
Part I: Cell lysate
(template) preparation
[1]
Select well isolated colonies
on LB plates for PCR amplification. Prepare a PCR tube for each colony.
[2]
Pick colony with a sterile
pipette tip and resuspend colony in 5μl of sterile
ddH2O.
[3]
Heat PCR tubes in the thermocycler at 98oC for 10 minutes to lyse the
cells. And centrifuge the lysate at 14,000 rpm for 1 minute.
Part
II: PCR
PCR Reaction Mixture
|
Volume of reaction mix (μl) for 1
reaction |
Volume of reaction mix (μl) for 10
reactions |
Quick-Load Taq
Master Mix (2X) |
7.5 |
75 |
VF2 primer (10 mM) |
0.075 |
0.75 |
VR primer (10 mM) |
0.075 |
0.75 |
ddH2O |
6.35 |
63.5 |
Total |
14 |
140 |
§ Mix 1 μl of DNA template (from step 3)
with 14 μl of master mix in a PCR tube.
Thermocycling Conditions
Step |
Temperature (oC) |
Time |
|
Initial denaturation |
95 |
30 seconds |
|
Denaturation |
95 |
30 s |
|
Annealing |
|
55 |
30 seconds |
Extension (This process is repeated for 32 cycles) |
68 |
5 minutes* |
|
Final extension |
68 |
5 minutes |
|
Hold |
4 |
∞ |
|
*Depending on the size of amplicon (e.g. 5 minutes for 4 to 5 kb)
</hr>
Genomic DNA Extraction Protocol
QIAGEN® DNeasy 96 Blood & Tissue Kit
[1]
Harvest cells by centrifugation
at 5000 x g (7500 rpm) for 10 minutes. Discard supernatant.
[2]
Resuspend cell pellet in 180 µl Buffer ATL.
[3]
Add 20 µl proteinase K to the
mixture. Mix thoroughly by vortex, and incubate in a 56°C water bath until the
tissue has completely lysed. Vortex occasionally during incubation.
[4]
Add 4 µl RNase A (100 mg/ml), mix by vortexing
and incubate at room temperature for 30 minutes.
[5]
Vortex for 15 seconds.
[6]
Add 200 µl of Buffer AL to the
sample, and mix thoroughly using a vortex.
[7]
Add 200 µl of ethanol
(96–100%), and mix again thoroughly by vortex.
[8]
Pipette the mixture from step 7
(including any precipitate) into the DNeasy Mini spin
column placed in a 2 ml collection tube. Centrifuge at 6000 x g (8000 rpm) for
1 minute. Discard the flow-through and collection tube.
[9]
Place the DNeasy
Mini spin column in a new 2 ml collection tube, add 500 µl of Buffer AW1, and
centrifuge for 1 minute at 6000 x g (8000 rpm). Discard the flow-through and
collection tube.
[10] Repeat step 9 with centrifuge time 3 minutes at 20,000 x g (14,000
rpm) to dry the DNeasy membrane. Discard the
flow-through and collection tube.
[11] Place the DNeasy Mini spin column in a
clean 1.5 ml microcentrifuge tube, and pipette 200 µl
of Buffer AE directly onto the DNeasy membrane.
Incubate at room temperature for 1 minute and then centrifuge for 1 min at 6000
x g (8000 rpm) to elute the DNA.
[12] Repeat the elution in step 11 to recover more DNA.
</hr>
Protein Extraction Protocol
BioVision® EZLysTM
Bacterial Protein Extraction Reagent
[1]
Collect
cells by centrifugation at 16,000g for 10 minutes in a pre-weighed centrifuge
tube. Remove as much liquid as possible. Determine the net weight of the cell
pellet.
[2]
Resuspend the pellet in at least 4 ml of EZLys reagent (pre-warmed to room temperature) per 1g of
wet cell paste by pipetting and/or gentle vortex.
[3]
Incubate
the cell suspension by shaking or slowly mixing for approximately 5 minutes.
[4]
Remove
the insoluble cell debris by centrifugation at 16,000g for 20 minutes at 4oC.
The supernatant containing the soluble proteins can now by analyzed or further
purified by using a Ni-NTA column or stored at -20oC for future use.
</hr>
ONPG Assay Protocol
[1] Incubate the E. coli culture in 10 ml of LB broth overnight at 37oC with shaking.
[2]
Measure the OD600 of
the overnight culture following a 10-fold dilution.
(Dilute culture with LB broth and use LB broth as blank)
[3] Transfer 1 ml of E. coli to a new glass test tube.
[4] Add 1 ml of Z buffer to the tube.
[5] Add 40 μl of chloroform and 20 μl of 0.1% SDS to the tube. Mix thoroughly.
[6] Incubate the tube in a 37oC water bath for 10 minutes. Mix vigorously.
[7] Add 0.4 ml of ONPG (4 mg/ml) to the tube and shake for a few times.
[8] Incubate the tube in a 37oC water bath for 15 minutes.
[9] Add 1 ml of 1 M Na2CO3 to the tube to stop the reaction.
[10] Transfer 1 ml of supernatant from the tube to a cuvette.
[11] Measure the OD420 of the supernatant.
(Dilute with LB broth if necessary and use 0.4 ml ONPG + 2 ml Z buffer + 1 ml
Na2CO3 as blank)
</hr>
RNA Extraction Protocol
Using TaKaRa® MiniBEST
Universal RNA Extraction Kit
[1] Harvest 500 μl of bacterial cells in a 1.5 ml microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 x g) for 1 minute at 4oC.
[2] Remove the supernatant. Resuspend the cell pellet in 200 μl of freshly prepared lysozyme (0.5 mg/mL in Tris/EDTA Buffer) solution and ensure no cell clumps can be seen. Incubate at room temperature for 15 minutes.
[3] Add 600 μl of Buffer RL and 12 μl of 50X DTT Solution. Mix thoroughly. Incubate the cell lysate at room temperature for 2 minutes.
[4] Apply the cell lysate to a gDNA Eraser spin column with a 2 ml collection tube. Centrifuge at 12,000 rpm (~13,400 x g) for 1 minute.
[5] Discard the gDNA Eraser Spin Column. Add an equal volume of 70% ethanol (812 μl) to the flow-through in the 2 ml collection tube. Mix thoroughly.
[6] Transfer the mixture to an RNA spin column with 2 ml collection tube. (If the volume of the mixture is more than 600 μl, apply the mixture by dividing into several aliquots). Centrifuge at 12,000 rpm (~13,400 x g) for 1 minute. Discard the flow-through.
[7] Add 500 μl Buffer RWA to the RNA spin column. Centrifuge at 12,000 (~13,400 x g) for 30 seconds. Discard the flow-through.
[8] Add 600 μl Buffer RWB to the RNA spin column. Centrifuge at 12,000 (~13,400 x g) for 30 seconds. Discard the flow-through.
[9] Prepare the DNase I mixture in a 1.5 ml microcentrifuge tube by adding 5 μl 10X DNase I Buffer, 4 μl Recombinant DNase and 41 μl RNase free H2O. Mix gently by inverting the tube.
[10] Add 50 μl of the DNase I mixture to the center of membrane. Incubate at room temperature for 15 minutes.
[11] Add 350 μl RWB to the RNA spin column. Centrifuge at 12,000 rpm (~13,400 x g) for 30 seconds. Discard the flow-through.
[12] Repeat step 8.
[13] Centrifuge the empty spin column at 12,000 rpm (~13,400 x g) for 2 minutes.
[14] Place the RNA spin column in a new 1.5 ml RNase free collection tube. Add 30 μl of RNase-free water to the center of membrane. Incubate at room temperature for 5 minutes. Centrifuge for 2 minutes at 12,000 rpm (~13,400 x g).
[15] Repeat step 14 by using the flow-through (RNA) in the 1.5 ml RNase free collection tube.
[16] Store the RNA at -80oC.
</hr>
Reverse-transcription PCR Protocol
Using TaKaRa PrimeScript™ RT reagent
Kit with gDNA Eraser
Part I: Removal of genomic DNA
in RNA samples
Digestion of genomic DNA
|
Volume (μl) |
Master mix (μl) |
gDNA Eraser Buffer (5X) |
2.0 |
16 |
gDNA Eraser |
1.0 |
8.0 |
Total |
3.0 |
24 |
§ Add 1 µg of each total RNA template to 3 μl of master mix in each PCR tube
§ Add an appropriate volume of RNase-free sterile dH2O to yield a final volume of 10 μl in each PCR tube
§ Place the tubes inside the thermocycler under 37oC for 2 minutes, followed by 85oC for 5 seconds.
Part II:
Reverse-transcription reaction
Reverse-transcription
reaction
|
Volume (μl) |
Master mix (μl) |
PrimeScript Buffer 2 (5X) |
4.0 |
32 |
PrimeScript RT Enzyme Mix I |
1.0 |
8.0 |
RT Primer Mix |
1.0 |
8.0 |
RNase Free dH2O |
4.0 |
32 |
Total |
10.0 |
80.0 |
§ Add 10.0 μl of reaction solution from step 1 to 10.0 μl of master mix in each PCR tube
§ Place the tubes inside the thermocycler under 37oC for 2 minutes, followed by 85oC for 5 seconds.
</hr>
Real time PCR Protocol
Using Promega GoTaq® qPCR Master Mix
Part
I: Standard Curve Construction
[1]
Prepare
4 concentrations of cDNA template by serially diluting a standard RT reaction
product as follows:
Dilution |
Recipe |
A (1/5) |
20 μl RT reaction product + 80 μl water |
B (1/50) |
10 μl Dilution A + 90 μl water |
C (1/500) |
10 μl Dilution B + 90 μl water |
D (1/5000) |
10 μl Dilution C + 90 μl water |
[2]
Add
2 μl
of each concentration (in triplicate) to appropriate wells on a 96-well PCR
plate:
|
Triplicate |
||
Conc. |
1 |
2 |
3 |
A |
○ |
○ |
○ |
B |
○ |
○ |
○ |
C |
○ |
○ |
○ |
D |
○ |
○ |
○ |
[3]
Prepare
a PCR master mix as follows:
|
Volume (μl) |
Master mix (μl) |
GoTaq® qPCR Master Mix (2X) |
5.0 |
80 |
SYBP Green Supermix |
0.1 |
1.3 |
10 μM Forward primer |
0.18 |
2.34 |
10 μM Reverse primer |
0.18 |
2.34 |
Water |
2.54 |
33.02 |
Diluted cDNA |
2.0 |
(pre-add to well) |
Total |
10 |
130 |
[4]
Mix
a 8 μl PCR master mix with each cDNA aliquot by gently pipetting up and
down several times
[5]
Seal the 96-well plate with an
optical tape. Spin down all droplets by centrifugation at 3,000 rpm for 2
minutes.
Part II: Gene expression
measurements
[1]
Dilute
RT product 1 in 5 times as follows:
à20 μl RT reaction product + 80 μl water
If the expression of the target gene is low, prepare a less diluted sample of
the RT-product for analysis.
[2]
Add
2 μl
of the diluted RT product (in triplicate) to appropriate wells of a 96-well PCR
plate.
[3]
Prepare
a PCR master mix as follows:
|
Volume (μl) |
Master mix (μl) |
GoTaq® qPCR Master Mix (2X) |
5.0 |
20 |
SYBP Green Supermix |
0.1 |
0.4 |
10 μM Forward primer |
0.18 |
0.72 |
10 μM Reverse primer |
0.18 |
0.72 |
Water |
2.54 |
10.16 |
Diluted cDNA |
2.0 |
(pre-add to well) |
Total |
10 |
40 |
[4]
Mix
8 μl
PCR master mix with each cDNA aliquot by gently pipetting up and down several
times
[5]
Seal the 96-well plate with an optical
tape. Spin down all droplets by centrifugation at 3,000 rpm for 2 minutes.