Difference between revisions of "Team:elan vital korea/Protocol"

Latest revision as of 12:14, 18 September 2015

WETLAB
-Protocol-

Protocols

We conducted our experiments by following the protocols below. As an official procedure, lab workers should understand the lab experiment assigned to them along with safety procedures before starting lab work. The protocols are arranged according to the order of experiments we followed.

How to handle reagents.

1. Reagents used in our project, such as restriction enzymes, must be stored in low temperature. The reagents must be stored in the freezer when they are not used, and must be put on ice when taking them out of the freezer for an experiment.

2. Reagents should be added last to the solution, because reagents are sensitive to inactivation by pH and ionic conditions that deviate from their storage and reaction buffers. After adding reagents, the mixed solution should be mixed completely.

Protocols to store materials and maintain
usage history of each material.

1. Reporter cell, test cell and competent cell (Top 10 invitrogen) must be kept at 4°C and frequently used reagents, reagents, DNA plasmids should be kept at −20°C in the freezer.

2. We use triple distilled water (or DDH2O) to make LB broth. Triple distilled water is kept at lab temperature (around 18 °C or lower).

3. Other materials such as yeast and NaCl are stored and maintained under the responsibility of Gachon Molecular Biology Lab.

4. We have to record the history of each material, including if plasmids/reporter cell/ test cell/ AHL have been frozen and if so, when it is used.

LB Medium

1. To prevent contamination, we only used LB medium made within three days.

2. Materials: Sodium Chloride (LB Media, Sigma), Trypton(LB Media, Sigma), Yeast Extract(LB Media Sigma), ddH2O (triple distilled water)

3. Equipment: autoclave, electronic scale.

4. Protocol For 200mL LB bottle
1) 2 g of Sodium Chloride to a final concentration of 0.17 M
2) 2g of 1%(w/v) Bacto™ tryptone
3) 1g of 0.5% (w/v) yeast extract
4) ddH2O to 200 mL
5) Autoclave for 20 min within 2 hours
6) Keep at room temperature

LB Agar Plates and Addition of Antibiotics

1. We have used LB (solidified lysogeny broth), rich growth medium for E.coli, in our experiments.

2. Just before pouring the solution into petri dishes, an antibiotic can be added for resistance selection. We followed the normal working concentrations such as:

- chloramphenicol: 25 μg/mL (Chloramphenicol stock is dissolved in ethanol) In case of using ampicillin: 100 μg/mL
- normal stock concentrations:1000-fold

3. Material to make LB plates:
Sodium Chloride (LB Media, Sigma) Bacto™ tryptone (LB Media, Sigma) yeast extract (LB Media, Sigma) Bacto™ agar (LB Media, Sigma) ddH2O (triple distilled water) 1000x chloramphenicol or ampicillin

4. LB agar preparation protocol
We usually make 1liter bottle for LB Agar
1) 200 mL LB prepared fresh, non-autoclaved
2) 3 g agar
3) Shake until all solids are dissolved
4) Autoclave for 20 min within 2 hr
5) Keep it cool until it reaches around 40-50 °C
6) Add 200 μL of 1000x chloramphenicol and gently stir it. Be careful not to shake the bottle too long/hard so that bubbles are created.
7) Pour into empty petri dishes just enough to cover the surface (~20 mL per plate). In case that bubbles are in the plate, heat the plate surface carefully with a burner only until the bubbles are burst but the solution is heated.
8) Leave the plates at room temperature around one hour until it is solidified.
9) Solidified plates should be turned upside down for a few hours at room temperature, then stored at 4°C.

Overnight Cultures with Antibiotics

1. We have conducted overnight culture for a single bacterial strain which process needs a plate or medium with single colonies and LB containing chloramphenicol.

2. Material Needed chloramphenicol: 25 μg/mL Normal stock concentrations: 1000-fold higher In case of using ampicillin: 100 μg/mL

3. Protocol
1) Quickly burn the neck of a bottle containing LB medium before pouring it out into a tube. Even the slightest contamination of LB will be damaging.
2) Add chloramphenicol or ampicillin to give the appropriate concentration
3) Scoop one colony from the plate with a sterile micropipette tip
4) Immediately stick the tip into the tube containing the medium and chloramphenicol or ampicillin
5) Incubate at 37°C with the shaking incubator overnight.

Agarose Gel Electrophoresis

1. Agarose gel electrophoresis is used for separation and analysis of larger (>100 bases in length) nucleic acids under non-denaturing conditions.

2. Analysis requires that the gel contains a DNA stain visible under UV light. Since the stain interacts with nucleic acids and is therefore potentially mutagenic, always wear nitrile gloves when working with agarose gels.

3. Use protective glasses when using the UV light box.

4. Material Needed
Agarose
1x TBE
Sybr®Safe
Loading dye mix
DNA ladder size marker
DNA samples

5. Protocol:
1) The gel tray must be on a level surface.
2) Insert the comb into the gel tray at one end ~1 cm from the edge.
3) For a 1% 150 mL agarose gel, weigh 1.5 g of agarose in a 500 mL conical flask.
4) Add 150 mL 1x TBE buffer.
5) To dissolve the agarose in the buffer, swirl to mix and microwave for a few minutes taking care not to boil the solution out of the flask.
6) Remove the flask occasionally and check whether the agarose has dissolved completely.
7) Let the agarose solution cool down.
8) Once the solution is touchable, add the DNA stain.
9) Check the stock concentration as the working concentration for ethidium bromide is 0.5 μg/mL while for Sybr®Safe it is simply 1x.
10) Pour the gel solution into the gel tray.
11) Remove any air bubbles with a pipette tip.
12) Put in comb.
13) The gel will solidify while cooling down to room temperature, which usually takes about 30 min.
14) Running the gel by the following procedure
a. Release the gel tray from the tape or casting stand.
b. Place the gel tray into the buffer chamber and remove the comb carefully
c. Add 1x TBE buffer until the gel is completely covered.
d. Take the DNA sample (~0.2 μg) and mix with loading dye.
e. Load the size marker mixed in 1x loading dye (~6 μL final volume) into a middle well.
f. Load the samples into the other wells while writing down which lanes have which samples.
g. Put the lid onto the buffer chamber and connect it to the power supply.
h. Run the gel at 100 V for 30–60 min. Neither of the two dyes should be run off the gel.
i. Stop the run and bring the gel to a UV table to visualize the gel bands.
j. Take a picture of the gel.

Gel Extraction

QIAquick®Gel Extraction Kit
Notes before starting

1. This protocol is for the purification of up to 10μg DNA (70bp to 10kb).

2. The yellow color of buffer QG indicates a pH ≤ 7.5. DNA adsorption to the membrane is only efficient at pH ≤ 7.5.

3. Add ethanol (96%100%) to Buffer PE before use (see bottle label for volume).

4. Isopropanol (100%) and a heating block or water bath at 50°C are required.

5. All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a convetional table-top microcentrifuge.

6. Symbosl: ● centrifuge processing; ▲ vacuum processing.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100μl). The maximum amount of gel per spin column is 400mg. For >2% agarose gels, add 6 volumes Buffer QG.

3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.

4. Add 1 volume isopropanol to the sample and mix.

5. Place a QIAquick spin column in ● a provided 2ml collection tube or into ▲ a vacuum amnifold. To bind DNA, aply the sample to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum to the manifold untill all the samples QIAquick column back into the same tube. For example volumes of 0> 800μl, load and spin/apply vacuum again.

6. If the DNA will subsequently be used for sequenceing, in vitro transcruption, or microinjection, add 500μl Buffer QG to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum. ● Discard flow through and place the QIAquick column back into the same tube.

7. To wash, add 750μl Bufick column and fer PE to QIAquickcolumn and ● centrifuge for 1 min or ▲ apply vacuum. ●Discard flow-through and place the QIAquick column back into the same tube.

8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

9. To elute DNA, add 50μl Buffer EB (10mM Tris•Cl, pH 8.5) or water to the center of the QIAquick memberane and centrifugethe colum for 1 min. For increased DNA concentration, add 30μl Buffer EB to the center of the QIAquick membrance, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrance, increasing the incubation time to up to 4 min can increase the yield of purified DNA.

10. If the Purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Transformation Procedure

Use this procedure to transform One Shot* TOP10 chemically competent E. coli. We recommend including the pUC19 control plasmid DNA supplied with the kit (10 pg/ μl in 5mM TrisHCl, 0.5mM EDTA, pH 8) in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.

1. Thaw, on ice, one vial of One Shot® TOP 10 chemically competent cells for each transformation.

2. Add 1 to 5 μl of the DNA (10pg to 100 ng) into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10pg (1μl) of DNA into a separate vial of One Shot® cells and mix gently.

3. Incubate the vial(s) on ice for 30 mins.

4. Heatshock the cells for 30 secs at 42°C without

5. Remove the vial(s) from the 42°C bath and place them on ice for 2 mins

6. Asceptically add 250 μl of prewarmed S.O.C. Medium to each vial.

7. Cap the vial(s) tightly and shake horizontally at 37°CC for 1 hour at 225 rpm in shaking incubator.

8. Spread 20200 μl from each transformation on a prewarmed selective plate and incubate overnight at 37°C. We recommend that you plate two different volumes to ensure that at least one plate will have wellspaced colonies. For the pUC19 control, dilute the transformation mix 1:10 into LB Medium (e.g. remove 100μl of the transformation mix and add to 900μl of LB Medium) and plate 25-100μl.

9. Store the remaining transformation mix at +4°C. Additional cells may be plated the next day, if desired.

10. Invert the selective plate(s) and incubate at 37°C

Reporter Cell Assay Protocol

1. Measure out 1ml of the cells into tubes.

2. Thaw out AHL on ice

3. Put 3ul of AHL in test cell

4. Wait 30 minutes

5. Add 1ml of reporter cell to the test cell

6. Wait 3 hours

7. Put 200ul of the mixture into a well plate

8. Put the well plate in the spectrometer to observe the results. (various independent variables such as time or the amounts of the chemical were varied in our different experiments.) (It is usually a good idea to have a control group with empty LB medium instead of the test cell running alongside the main experiment.)

Mini and Midi preparation

QIAGEN® Plasmid Mini and Midi Kits
Notes before starting

1. Add RNase A solution to Buffer P1, mix, and store at 2-8°C

2. Optional: Add LyseBlue® reagent to Buffer P1 at a ratio of 1:1000.

3. Prechill Buffer P3 at 4°C. Check Buffer P2 for SDS precipitation

4. Isopropanol and 70% ethanol are required.

5. Symbols: ● QIAGEN Plasmid Mini Kit; ■ QIAGEN Plasmid Midi Kit

1. Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 mins at 4°C.

2. Resuspend the bacterial pellet in ● 0.3ml or ■ 4ml Buffer P1.

3. Add ● 0.3ml or ■ 4ml Buffer P2, mix thoroughly by vigorously inverting 4-6 times, and incubate at room temperature (1525°C) for 5 mins. If using LyseBlue reagent, the solution will turn blue.

4. Add ● 0.3ml or ■ 4 ml prechilled Buffer P3, mix thoroughly by vigorously inverting 4-6times. Incubate on ice for ● 5 mins or ■ 15 mins. If using LyseBlue reagent, mix the solution until it is colorless.

5. ●: Centrifuge at 14,000-18,000 x g for 10 mins at 4°C. Re-centrifuge if supernatant is not clear ■: Centrifuge at 20,000 x g for 30 mins at 4°C. Re-centrifuge the super natant at 20,000 x g for 15 mins at 4°C

6. Equlibriate a QIAGEN tip ● 20 or ■ 100 by applying ● 1ml or ■ 4ml Buffer QBT, and allow column to empty by gravity flow.

7. Apply these supernatant from step 5 to the QIAGEN tip and allow it to enter the resin by gravity flow.

8. Was the QIAGEN top with ● 2 x 2 ml or ■ 2 x10 ml Buffer QC. Allow Buffer QC to move through the QIAGEN top by gravity flow.

9. Elute DNA with ● 0.8 ml or ■ 5 ml Buffer QF into a clean ● 2 ml or ■ 15 ml vessel. For constructs larger than 45 kb, prewarming the elution buffer to 65°C may help to increase the yield.

10. Precipitate DNA by adding ● 0.56 ml or ■ 3.5 ml room temperature isopropanol to the eluted DNA and mix. Centrifuge at 15,000 x g for 30 mins at 4°C. Carefully decant the supernatant.

11. Wash the DNA pellet with ● 1 ml or ■ 2ml room temperature 70% ethanol and centrifuge at 15,000 x g for 10 mins. Carefully decant the supernatant.

12. Air dry pellet for 5-10 mins and redissolve DNA in a suitable volume of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM TrisCl, pH 8.5).

Ligation

Quick Ligation Protocol

1. Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10μl with dH2O.
2. Add 10μl of 2X Quick Ligation Reaction Bugger and mix.
3. Add 1μl of Quick T4 DNA Ligase and mix thoroughly.
4. Centrifuge briefly and incubate at room temperature (25°C) for 5mins.
5. Chill on ice, then transform or store at -20°C
6. Do not heat inactivate. Heat activation dramatically reduces transformation efficiency.

Enzyme Digestion

Plasmid Construction Protocol

Preliminary Work

Material

Lab Work for Plasmid Construction

Control Experiment

First, let's see a video showing our control experiment work:

Purpose: investigate the concentration of AHL;
Find out how much AHL concentration is optimal and how much time it takes for the AHL to express GFP;
Find out how much time it takes for the test cell to break down AHL
Protocol:
1. Incubate bacteria overnight at 37C
2. Separate out 1ml of test cell and 1ml of reporter cell
3. Add 3ul of AHL to the test cell
4. Incubate for 30 minutes
5. Add the reporter to the test cell
6. Incubate for 3 hours
7. Move 200ul of the solution into a well plate
8. Put the well plate into the spectronometer
9. View the results

@@ Line 62: / Line 62: @@
 #home {
-     background: url(https://static.igem.org/mediawiki/2015/6/66/HumanPracticeBG.png) 50% 0 no-repeat fixed;
+     background: url(https://static.igem.org/mediawiki/2015/b/b3/Bg2.png) 50% 0 no-repeat fixed;
 min-height:1000px;
      height: 1000px;
@@ Line 72: / Line 72: @@
 #about {
 background: url(https://static.igem.org/mediawiki/2015/9/9c/Newlightbg.jpg) 50% 0 no-repeat fixed;
-    min-height:1000px;
+/*    min-height:1000px;
-height: 1000px;
+height: 1000px; */
+height: auto;
      margin: 0 auto;
      width: 100%;
@@ Line 81: / Line 82: @@
 #maintext {
 background: url(https://static.igem.org/mediawiki/2015/9/9c/Newlightbg.jpg) 50% 0 no-repeat fixed;
-    height: 1000px;
+/*    height: 1000px; */
 min-height:1000px;
      margin: 0 auto;
@@ Line 91: / Line 92: @@
 #maintext2 {
 background: url(https://static.igem.org/mediawiki/2015/9/9c/Newlightbg.jpg) 50% 0 no-repeat fixed;
-    height: 1000px;
+/*    height: 1000px; */
 min-height:1000px;
      margin: 0 auto;
@@ Line 176: / Line 177: @@
 p {
-font-size:16px;
+font-size:18px;
 line-height:170%;
 }
@@ Line 228: / Line 229: @@
 .inner1 {
 width: 30%;
-height: auto;
+/* height: auto; */
 margin-left:35%;
+margin-bottom: 10px;
 }
@@ Line 324: / Line 326: @@
      <!-- Section #2 --!>
-         <section id="about" data-speed="10" data-type="background">
+         <section id="about" data-speed="10" data-type="background" >
       <h5 style="text-align:center;">
-<font color:black;>
+ <font color="black">
 <a name="myAnchor" id="myAnchor"></a><br><br><br>Protocols
 </font>
@@ Line 340: / Line 342: @@
 </p>
 </div>
+</section>
-<br><Br>
+ <section id="maintext0" data-speed="10" data-type="background">
   <h5 style="text-align:center;">
   <font color="black">
@@ Line 353: / Line 356: @@
   <font color="black">
 <b>1.</b>
-Reagents used in our project, such as restriction reagents, must be stored in low temperature.  The reagents must be stored in the freezer when they are not used, and must be put on ice when taking them out of the freezer for an experiment. <br><br>
+Reagents used in our project, such as restriction enzymes, must be stored in low temperature.  The reagents must be stored in the freezer when they are not used, and must be put on ice when taking them out of the freezer for an experiment. <br><br>
 <b>2.</b>
@@ Line 361: / Line 364: @@
 </div>
-<br><br>
+<br>
   <h5 style="text-align:center;">
   <font color="black">
@@ Line 388: / Line 391: @@
 </div>
-<a href="#top" rel="" id="top" class="anchorLink"><img class="displayed" src="https://static.igem.org/mediawiki/2015/5/5b/Scroll_arrow_top_Black.png"></a>
- <h6 style="text-align:center;"> <font color="black">
-                   To The Top
-               </font> </h6>
-        </section>
-    <!-- Section #3 --!>
-        <section id="maintext" data-speed="10" data-type="background">
-<br><Br>
   <h5 style="text-align:center;">
   <font color="black">
@@ Line 410: / Line 401: @@
 <P style="text-align:left;">
   <font color="black">
-<b>1.</b> To prevent contamination, we only used LB medium made within three days. <br>
+<b>1.</b> To prevent contamination, we only used LB medium made within three days. <br><br>
-<b>2.</b> Materials: Sodium Chloride (LB Media, Sigma), Trypton(LB Media, Sigma), Yeast Extract(LB Media Sigma), ddH2O (triple distilled water) <br>
+<b>2.</b> Materials: Sodium Chloride (LB Media, Sigma), Trypton(LB Media, Sigma), Yeast Extract(LB Media Sigma), ddH2O (triple distilled water) <br><br>
-<b>3.</b> Equipment: autoclave, electronic scale. <br>
+<b>3.</b> Equipment: autoclave, electronic scale. <br><br>
 <b>4.</b> Protocol For 200mL LB bottle <br>
 ) 2 g of Sodium Chloride to a final concentration of 0.17 M  <br>
@@ Line 424: / Line 415: @@
 </div>
+<br><Br>
   <h5 style="text-align:center;">
   <font color="black">
@@ Line 430: / Line 421: @@
 </font>
 </h5>
+<br>
@@ Line 435: / Line 427: @@
 <P style="text-align:left;">
   <font color="black">
-<b>1.</b> We have used LB (solidified lysogeny broth), rich growth medium for E.coli, in our experiments. <br>
+<b>1.</b> We have used LB (solidified lysogeny broth), rich growth medium for E.coli, in our experiments. <br><br>
-<b>2.</b> Just before pouring the solution into petri dishes, an antibiotic can be added for resistance selection. We followed the normal working concentrations such as: <br>
+<b>2.</b> Just before pouring the solution into petri dishes, an antibiotic can be added for resistance selection. We followed the normal working concentrations such as: <br /><br />
 - chloramphenicol: 25 μg/mL (Chloramphenicol stock is dissolved in ethanol) In case of using ampicillin: 100 μg/mL <br>
-- normal stock concentrations:1000-fold   <br>
+- normal stock concentrations:1000-fold   <br><br>
-<b>3.</b> Material to make LB plates: Sodium Chloride (LB Media, Sigma) Bacto™ tryptone (LB Media, Sigma) yeast extract (LB Media, Sigma) Bacto™ agar (LB Media, Sigma) ddH2O (triple distilled water) 1000x chloramphenicol or ampicillin <br>
+<b>3.</b> Material to make LB plates: <br /> Sodium Chloride (LB Media, Sigma) Bacto™ tryptone (LB Media, Sigma) yeast extract (LB Media, Sigma) Bacto™ agar (LB Media, Sigma) ddH2O (triple distilled water) 1000x chloramphenicol or ampicillin <br><br>
-<b>4.</b> LB agar preparation protocol We usually make 1liter bottle for LB Agar <br>
+<b>4.</b> LB agar preparation protocol <br /> We usually make 1liter bottle for LB Agar <br>
 ) 200 mL LB prepared fresh, non-autoclaved <br>
 ) 3 g agar <br>
@@ Line 462: / Line 454: @@
      <!-- Section #4 --!>
          <section id="maintext2" data-speed="10" data-type="background">
+<br><br>
+ <h5 style="text-align:center;">
+ <font color="black">
+Overnight Cultures with Antibiotics
+</font>
+</h5>
 <br>
-        </section>
+<div class="inner1">
+<P style="text-align:left;">
+ <font color="black">
+<b>1.</b> We have conducted overnight culture for a single bacterial strain which process needs a plate or medium with single colonies and LB containing chloramphenicol. <br><br>
+<b>2.</b> Material Needed chloramphenicol: 25 μg/mL Normal stock concentrations: 1000-fold higher In case of using ampicillin: 100 μg/mL <br><br>
+<b>3.</b> Protocol<br>
+) Quickly burn the neck of a bottle containing LB medium before pouring it out into a tube. Even the slightest contamination of LB will be damaging. <br>
+) Add chloramphenicol or ampicillin to give the appropriate concentration<br>
+) Scoop one colony from the plate with a sterile micropipette tip<br>
+) Immediately stick the tip into the tube containing the medium and chloramphenicol or ampicillin<br>
+) Incubate at 37°C with the shaking incubator overnight.<br>
+</font>
+</p>
+</div>
+<br><br>
+ <h5 style="text-align:center;">
+ <font color="black">
-    <!-- Section #5 --!>
+Agarose Gel Electrophoresis
-        <section id="maintext" data-speed="10" data-type="background">
+</font>
+</h5>
 <br>
-<div class="inner3">
-<font color="black">
-   <div class="caption4">
-  <h5>
- <font color="black"> 19 </font>
-    </h5>
-         <p>
-Inflammable chemicals including acetone, oil, or gas must be kept in the place where ventilation is easy and access is infrequent.
-         </p>
-   </div>
-   <div class="caption4">
+<div class="inner1">
-  <h5>
+<P style="text-align:left;">
-  <font color="black"> 20 </font>
+  <font color="black">
-    </h5>
-         <p>
-Importing and exporting of high-pressure gas container must be done using transportation equipment.
-         </p>
-   </div>
+<b>1.</b> Agarose gel electrophoresis is used for separation and analysis of larger (>100 bases in length) nucleic acids under non-denaturing conditions. <br><br>
-   <div class="caption4">
+<b>2.</b> Analysis requires that the gel contains a DNA stain visible under UV light. Since the stain interacts with nucleic acids and is therefore potentially mutagenic, always wear nitrile gloves when working with agarose gels.<br><br>
-  <h5>
+<b>3.</b> Use protective glasses when using the UV light box.<br><br>
- <font color="black"> 21 </font>
+<b>4.</b> Material Needed<br>
-    </h5>
+Agarose<br>
-         <p>
+x TBE<br>
-When storing high-pressure gas containers, they must be safely and firmly fixed, isolated from flammable substances and inflammatory materials.
+Sybr®Safe<br>
-         </p>
+Loading dye mix<br>
-   </div>
+DNA ladder size marker<br>
+DNA samples<br><br>
-   <div class="caption4">
+<b>5.</b> Protocol: <br>
-  <h5>
+) The gel tray must be on a level surface.<br>
- <font color="black"> 21 </font>
+) Insert the comb into the gel tray at one end ~1 cm from the edge.<br>
-    </h5>
+) For a 1% 150 mL agarose gel, weigh 1.5 g of agarose in a 500 mL conical flask. <br>
-         <p>
+) Add 150 mL 1x TBE buffer. <br>
-Experiments using flammable, explosive, toxic, or volatile gases or vapors must be carried out in a hood.
+) To dissolve the agarose in the buffer, swirl to mix and microwave for a few minutes taking care not to boil the solution out of the flask. <br>
-         </p>
+) Remove the flask occasionally and check whether the agarose has dissolved completely. <br>
-   </div>
+) Let the agarose solution cool down.<br>
+) Once the solution is touchable, add the DNA stain. <br>
-   <div class="caption4">
+) Check the stock concentration as the working concentration for ethidium bromide is 0.5 μg/mL while for Sybr®Safe it is simply 1x.<br>
-  <h5>
+) Pour the gel solution into the gel tray. <br>
- <font color="black"> 22 </font>
+) Remove any air bubbles with a pipette tip. <br>
-    </h5>
+) Put in comb.<br>
-         <p>
+) The gel will solidify while cooling down to room temperature, which usually takes about 30 min.<br>
-Reagents container or cabinets for storing reagents must be kept in cool, well-ventilated places without direct sunlight, and far away from fire and heat sources.
+) Running the gel by the following procedure <br>
-         </p>
+a. Release the gel tray from the tape or casting stand. <br>
-   </div>
+b. Place the gel tray into the buffer chamber and remove the comb carefully<br>
+c. Add 1x TBE buffer until the gel is completely covered.<br>
-   <div class="caption4">
+d. Take the DNA sample (~0.2 μg) and mix with loading dye. <br>
-  <h5>
+e. Load the size marker mixed in 1x loading dye (~6 μL final volume) into a middle well.<br>
- <font color="black"> 23 </font>
+f. Load the samples into the other wells while writing down which lanes have which samples.<br>
-    </h5>
+g. Put the lid onto the buffer chamber and connect it to the power supply.<br>
-         <p>
+h. Run the gel at 100 V for 30–60 min. Neither of the two dyes should be run off the gel. <br>
-Experimental drugs shall be stored in a way not to be shaken by outside shock, and so that its storage bottles do not fall
+i. Stop the run and bring the gel to a UV table to visualize the gel bands. <br>
-         </p>
+j. Take a picture of the gel.<br>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 24 </font>
-    </h5>
-         <p>
-Toxic materials shall be handled using protective films, or other safety tents to prevent damages from splatter, heating or explosion so as to ensure the safety of the experimenter.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 25 </font>
-    </h5>
-         <p>
-Containers for chemical wastes shall not be left in the hallway or on the stairs, and shall not be stored in the corners of the laboratory or other invisible places.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 26 </font>
-    </h5>
-         <p>
-<font color="black">
-When performing microbiological tests, gloves shall be worn, and after the experiment, the used glassware shall be
-washed through sterilization, and disposable supplies and medium shall be disposed of separately from general garbage.
-</font>
-         </p>
-   </div>
 </font>
+</p>
 </div>
          </section>
-   <!-- Section #6 --!>
-        <section id="maintext2" data-speed="10" data-type="background">
-<br>
-<div class="inner4">
- <font color="black">
-   <div class="caption3">
-  <h5>
- <font color="black"> 27 </font>
-    </h5>
-         <p>
-Inflammable chemicals including acetone, oil, or gas must be kept in the place where ventilation is easy and access is infrequent.
-         </p>
-   </div>
-   <div class="caption3">
-  <h5>
- <font color="black"> 28 </font>
-    </h5>
-         <p>
-Importing and exporting of high-pressure gas container must be done using transportation equipment.
-         </p>
-   </div>
-   <div class="caption3">
-  <h5>
- <font color="black"> 29 </font>
+     <!-- Section #5 --!>
-     </h5>
+        <section id="maintext" data-speed="10" data-type="background">
-         <p>
-When storing high-pressure gas containers, they must be safely and firmly fixed, isolated from flammable substances and inflammatory materials.
-         </p>
-   </div>
-</font>
-</div>
- <h5 style="text-align:center;">
- <font color="black">
-Other significant Safety Considerations.
-</font>
-</h5>
 <br><br>
   <h5 style="text-align:center;">
   <font color="black">
-. Fire Regulations.
+Gel Extraction <br>
 </font>
 </h5>
+<br>
-<P style="text-align:center;">
+<div class="inner1">
+<P style="text-align:left;">
   <font color="black">
-When fire breaks out, the following guidelines shall apply.
+QIAquick®Gel Extraction Kit <br>
+Notes before starting<br /><br />
+<b>1.</b> This protocol is for the purification of up to 10μg DNA (70bp to 10kb). <br><br>
+<b>2.</b> The yellow color of buffer QG indicates a pH ≤ 7.5. DNA adsorption to the membrane is only efficient at pH ≤ 7.5.<br><br>
+<b>3.</b> Add ethanol (96%100%) to Buffer PE before use (see bottle label for volume). <br><br>
+<b>4.</b> Isopropanol (100%) and a heating block or water bath at 50°C are required. <br><br>
+<b>5.</b> All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a convetional table-top microcentrifuge. <br><br>
+<b>6.</b> Symbosl: ● centrifuge processing; ▲ vacuum processing. <br><br />
+<b>1.</b>  Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. <br>
+<b>2.</b>  Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100μl). The maximum amount of gel per spin column is 400mg. For >2% agarose gels, add 6 volumes Buffer QG. <br>
 </font>
-</p>
+</h5>
+<br>
+        </section>
-<div class="inner3">
- <font color="black">
-   <div class="caption6">
-  <h5>
- <font color="black"> 1) </font>
-    </h5>
-         <p>
-Save lives.
-         </p>
-   </div>
-    <div class="caption6">
+    <!-- Section #6 --!>
-  <h5>
+        <section id="maintext2" data-speed="10" data-type="background">
- <font color="black"> 2) </font>
-    </h5>
-         <p>
-Call the fire brigade.
-         </p>
-   </div>
-   <div class="caption6">
-  <h5>
- <font color="black"> 3) </font>
-    </h5>
-         <p>
-Alert people in the area.
-         </p>
-   </div>
+<div class="inner1">
-   <div class="caption6">
+<P style="text-align:left;">
-  <h5>
+  <font color="black">
+<b>3.</b>  Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow. <br><br />
-  <font color="black"> 4) </font>
+<b>4.</b>  Add 1 volume isopropanol to the sample and mix. <br><br />
-    </h5>
+<b>5.</b>  Place a QIAquick spin column in ● a provided 2ml collection tube or into ▲ a vacuum amnifold. To bind DNA, aply the sample to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum to the manifold untill all the
-         <p>
+samples QIAquick column back into the same tube. For example volumes of 0> 800μl, load and spin/apply vacuum again. <br><br />
-Extinguish the fire if possible.
+<b>6.</b>  If the DNA will subsequently be used for sequenceing, in vitro transcruption, or microinjection, add 500μl Buffer QG to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum. ● Discard flow through and place the
-         </p>
+QIAquick column back into the same tube. <br><br>
-   </div>
+<b>7.</b> To wash, add 750μl Bufick column and fer PE to QIAquickcolumn and ●
+centrifuge for 1 min or ▲ apply vacuum. ●Discard flow-through and place the
-   <div class="caption6">
+QIAquick column back into the same tube. <br><br>
-  <h5>
+<b>8.</b> Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
-  <font color="black"> 5) </font>
-    </h5>
-         <p>
-Close doors to the area.
-         </p>
-   </div>
-   <div class="caption6">
-  <h5>
- <font color="black"> 6) </font>
-    </h5>
-         <p>
-Evacuate.
-         </p>
-   </div>
-   <div class="caption6">
-  <h5>
- <font color="black"> 7) </font>
-    </h5>
-         <p>
-Reassemble outside the building at the designated meeting point.
-         </p>
-   </div>
 </font>
-</div>
+</h5>
          </section>
@@ Line 722: / Line 603: @@
      <!-- Section #7 --!>
+<div class="inner1">
+<P style="text-align:left;">
+ <font color="black">
+<b>9.</b> To elute DNA, add 50μl Buffer EB (10mM Tris•Cl, pH 8.5) or water to the
+center of the QIAquick memberane and centrifugethe colum for 1 min. For
+increased DNA concentration, add 30μl Buffer EB to the center of the
+QIAquick membrance, let the column stand for 1 min, and then centrifuge for
+min. After the addition of Buffer EB to the QIAquick membrance, increasing
+the incubation time to up to 4 min can increase the yield of purified DNA. <br><br>
+<b>10.</b> If the Purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye
+to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
+</font>
+</h5>
+<br>
+</div>
-        <section id="maintext" data-speed="10" data-type="background">
+<br><br>
+ <h5 style="text-align:center;">
+ <font color="black">
+Transformation Procedure
+</font>
+</h5>
 <br>
-<P style="text-align:center;">
+<div class="inner0">
-<font color="black">
+<P style="text-align:left;">
-Also it is very important to know the locations of the fire-fighting equipment, fire alarms, and evacuation routes closest to the lab. <br>
+ <font color="black">
-A small fire can be extinguished quickly by smothering it in a fire blanket or by spraying it with a fire extinguisher. <br>
+Use this procedure to transform One Shot* TOP10 chemically competent E. coli. We recommend including the pUC19 control plasmid DNA supplied with the kit (10 pg/ μl in 5mM TrisHCl, 0.5mM EDTA, pH 8) in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.
-Lab fires in biological labs are caused most commonly by the plating of cell cultures. <br>
-To prevent this, <br><br>
 </font>
 </p>
+</div>
+<br>
+</section>
-<div class="inner3">
-<font color="black">
-   <div class="caption4">
-  <h5>
- <font color="black"> 1 </font>
-    </h5>
-         <p>
-Never use paper bench coat near burners.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 2 </font>
-    </h5>
-         <p>
-Use a low reservoir volume of ethanol.
-         </p>
-   </div>
-   <div class="caption4">
+    <!-- Section #8 --!>
-  <h5>
+        <section id="maintext2" data-speed="10" data-type="background">
- <font color="black"> 3 </font>
-    </h5>
-         <p>
-Place the ethanol reservoir at least a foot from the burner and on the opposite side of the burner from the plates
-         </p>
-   </div>
-   <div class="caption4">
+<div class="inner1">
-  <h5>
+<P style="text-align:left;">
-  <font color="black"> 4 </font>
+  <font color="black">
-    </h5>
+<b>1.</b> Thaw, on ice, one vial of One Shot® TOP 10 chemically competent cells for each transformation. <br><br>
-         <p>
+<b>2.</b> Add 1 to 5 μl of the DNA (10pg to 100 ng) into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10pg (1μl) of DNA into a separate vial of One Shot® cells and mix gently. <br><br>
-Never wear plastic gloves when working with a flame.
+<b>3.</b> Incubate the vial(s) on ice for 30 mins. <br><br>
-         </p>
+<b>4.</b> Heatshock the cells for 30 secs at 42°C without <br><br>
-   </div>
+<b>5.</b> Remove the vial(s) from the 42°C bath and place them on ice for 2 mins <br><br>
+<b>6.</b> Asceptically add 250 μl of prewarmed S.O.C. Medium to each vial. <br><br>
-   <div class="caption4">
+<b>7.</b> Cap the vial(s) tightly and shake horizontally at 37°CC for 1 hour at 225 rpm
-  <h5>
+in shaking incubator. <br><br>
- <font color="black"> 5 </font>
+<b>8.</b> Spread 20200 μl from each transformation on a prewarmed selective plate
-    </h5>
+and incubate overnight at 37°C. We recommend that you plate two different
-         <p>
+volumes to ensure that at least one plate will have wellspaced colonies. For
-Carry the gas container for the burner carefully with a firm grip. If it is dropped, make sure that there is no leakage by smelling for gas.
+the pUC19 control, dilute the transformation mix 1:10 into LB Medium (e.g.
-         </p>
+remove 100μl of the transformation mix and add to 900μl of LB Medium) and
-   </div>
+plate 25-100μl. <br><br>
+<b>9.</b> Store the remaining transformation mix at +4°C. Additional cells may be
-   <div class="caption4">
+plated the next day, if desired. <br><br>
-  <h5>
+<b>10.</b> Invert the selective plate(s) and incubate at 37°C <br><br>
- <font color="black"> 6 </font>
-    </h5>
-         <p>
-Notify the instructor of any gas leakage and do not light burners when there is a smell of gas.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 7 </font>
-    </h5>
-         <p>
-Always light the match before opening the gas valve.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 8 </font>
-    </h5>
-         <p>
-Avoid placing burners too close to overhanging shelves.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 9 </font>
-    </h5>
-         <p>
-Never leave the table while a burner is on.
-         </p>
-   </div>
 </font>
+</p>
 </div>
-        </section>
+       </section>
-     <!-- Section #8 --!>
+     <!-- Section #9 --!>
-         <section id="maintext2" data-speed="10" data-type="background">
+         <section id="maintext" data-speed="10" data-type="background">
-<br>
   <h5 style="text-align:center;">
   <font color="black">
-CHEMICALS
+Reporter Cell Assay Protocol
 </font>
 </h5>
 <br>
-<P style="text-align:center;">
+<div class="inner1">
+<P style="text-align:left;">
   <font color="black">
-On each chemical container, there is a label that specifies the potential danger of the substance for humans and/or the environment. <br>
+<b>1.</b> Measure out 1ml of the cells into tubes. <br><br>
-Chemicals should be handled cautiously with gloves, both for your safety and for decreasing the contamination risk.<br>
+<b>2.</b> Thaw out AHL on ice <br><br>
-Always wear a lab coat and shoes as additional protection. Read the signs on the chemical container and the Material Safety Data Sheet<br>
+<b>3.</b> Put 3ul of AHL in test cell <br><br>
-(MSDS; available online) for further direction.<br>
+<b>4.</b> Wait 30 minutes <br><br>
+<b>5.</b> Add 1ml of reporter cell to the test cell <br><br>
+<b>6.</b> Wait 3 hours <br><br>
+<b>7.</b> Put 200ul of the mixture into a well plate <br><br>
+<b>8.</b> Put the well plate in the spectrometer to observe the results. (various independent variables such as time or the amounts of the chemical were varied in our different experiments.) (It is usually a good idea to have a control group with empty LB medium  instead of the test cell running alongside the main experiment.)
 </font>
 </p>
+</div>
 <br>
   <h5 style="text-align:center;">
   <font color="black">
-BIO- SAFETY AND DISPOSAL
+Mini and Midi preparation <br>
 </font>
 </h5>
 <br>
+        </section>
-<P style="text-align:center;">
+    <!-- Section #9 -->
+        <section id="maintext2" data-speed="10" data-type="background">
+<div class="inner1">
+<P style="text-align:left;">
   <font color="black">
-When you are working to generate new organisms by molecular methods, you are regulated in most countries <br>
+QIAGEN® Plasmid Mini and Midi Kits <br>
-according to international biosafety guidelines:
+Notes before starting<br /><br />
-</font>
+<b>1.</b> Add RNase A solution to Buffer P1, mix, and store at 2-8°C <br><br>
-</p>
+<b>2.</b> Optional: Add LyseBlue® reagent to Buffer P1 at a ratio of 1:1000. <br><br>
+<b>3.</b> Prechill Buffer P3 at 4°C. Check Buffer P2 for SDS precipitation <br><br>
-<div class="inner">
+<b>4.</b> Isopropanol and 70% ethanol are required. <br><br>
- <font color="black">
+<b>5.</b> Symbols: ● QIAGEN Plasmid Mini Kit; ■ QIAGEN Plasmid Midi Kit <br><br><br><br>
-   <div class="caption2">
-  <h5>
- <font color="black"> 1 </font>
-    </h5>
-         <p>
-CDC, Atlanta. Biosafety in Microbiological and Biomedical Laboratories. <br>
-<a>http://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf </a>
-         </p>
-   </div>
-   <div class="caption2">
+<b>1.</b> Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 mins at
-  <h5>
+°C. <br><br>
- <font color="black"> 2</font>
+<b>2.</b> Resuspend the bacterial pellet in ● 0.3ml or ■ 4ml Buffer P1. <br><br>
-    </h5>
-         <p>
-WHO, Geneva. Laboratory Biosafety Manual, 2004.<br>
-<a>http://www.who.int/csr/resources/publications/ biosafety/Biosafety7.pdf</a>
-         </p>
-   </div>
-</font>
-</div>
+<b>3.</b> Add ● 0.3ml or ■ 4ml Buffer P2, mix thoroughly by vigorously inverting 4-6
+times, and incubate at room temperature (1525°C) for 5 mins. If using
+LyseBlue reagent, the solution will turn blue. <br><br>
-<div class="inner">
+<b>4.</b> Add ● 0.3ml or ■ 4 ml prechilled Buffer P3, mix thoroughly by vigorously
+inverting 4-6times. Incubate on ice for ● 5 mins or ■ 15 mins. If using
+LyseBlue reagent, mix the solution until it is colorless. <br><br>
- <font color="black">
+<b>5.</b> ●: Centrifuge at 14,000-18,000 x g for 10 mins at 4°C. Re-centrifuge if
-   <div class="caption2">
+supernatant is not clear
+■: Centrifuge at 20,000 x g for 30 mins at 4°C. Re-centrifuge the super
+natant at 20,000 x g for 15 mins at 4°C  <br><br>
-  <h5>
+<b>6.</b> Equlibriate a QIAGEN tip ● 20 or ■ 100 by applying ● 1ml or ■ 4ml Buffer QBT,
+and allow column to empty by gravity flow. <br><br>
- <font color="black"> 3 </font>
+<b>7.</b> Apply these supernatant from step 5 to the QIAGEN tip and allow it to enter
-    </h5>
+the resin by gravity flow. <br><br>
-         <p>
-ECDC, Directive 2000/54/ec of the European parliament and of the Council of 18 September 2000 on the protection of  workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/391/EEC. <br>
-<a>http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2000:262:0021:0045:EN:PDF </a>
-         </p>
-   </div>
-   <div class="caption2">
+<b>8.</b> Was the QIAGEN top with ● 2 x 2 ml or ■ 2 x10 ml Buffer QC. Allow Buffer QC
-  <h5>
+to move through the QIAGEN top by gravity flow. <br><br>
- <font color="black"> 4 </font>
+<b>9.</b> Elute DNA with ● 0.8 ml or ■ 5 ml Buffer QF into a clean ● 2 ml or ■ 15 ml
-    </h5>
+vessel. For constructs larger than 45 kb, prewarming the elution buffer to
-         <p>
+°C may help to increase the yield. <br><br>
-Institutional Bio-safety Committee, under the ministry of Health and Welfare, Korea Center for Disease Control & Prevention. <br>
-<a>http://biosafety.cdc.go.kr</a>
-         </p>
-   </div>
-</font>
-</div>
-        </section>
+<b>10.</b> Precipitate DNA by adding ● 0.56 ml or ■ 3.5 ml room temperature
+isopropanol to the eluted DNA and mix. Centrifuge at 15,000 x g for 30 mins
+at 4°C. Carefully decant the supernatant. <br><br>
+<b>11.</b> Wash the DNA pellet with ● 1 ml or ■ 2ml room temperature 70% ethanol
+and centrifuge at 15,000 x g for 10 mins. Carefully decant the supernatant. <br><br>
-    <!-- Section #9 --!>
+<b>12.</b> Air dry pellet for 5-10 mins and redissolve DNA in a suitable volume of  appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM TrisCl, pH 8.5). <br><br>
-        <section id="maintext" data-speed="10" data-type="background">
-<br><br><br>
-<P style="text-align:center;">
-<font color="black">
-When working with microorganisms such as bacteria and viruses, there are four BioSafety Levels (BSL) numbered BSL1–4.<br>
-Biosafety level is the level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed <br>
-The levels of containment range from the lowest biosafety level 1 to the highest at level 4, and Ministry of Health and Welfare and Korea Centers <br>
-for Disease Control and Prevention have specified these levels facility. <br>
-Biocontainment can be classified by the relative danger to the surrounding environment as biological <br>
-safety levels.  As of 2006, there are four safety levels. These are called BSL1 through BSL4 <br>
 </font>
 </p>
-<br>
+</div>
+       </section>
- <h5 style="text-align:center;">
-                <font color="black">
-BSL 1
-</font>
-</h5>
-<br>
-<div class="inner3">
-<font color="black">
-   <div class="caption4">
-  <h5>
- <font color="black"> 1 </font>
-    </h5>
-         <p>
-involving well-characterized agents not known to consistently cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 2 </font>
-    </h5>
-         <p>
-bacteria and viruses including canine hepatitis, non-pathogenic Escherichia coli, as well as cell cultures and  non-infectious bacteria.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 3 </font>
-    </h5>
-         <p>
-The laboratory is not necessarily separated from the general traffic patterns in the building.
-         </p>
-   </div>
-   <div class="caption7">
+    <!-- Section #10 --!>
-  <h5>
+        <section id="maintext" data-speed="10" data-type="background">
- <font color="black"> 4 </font>
-    </h5>
-         <p>
-Conducted on open bench tops using standard microbiological practices.
-         </p>
-   </div>
-   <div class="caption7">
-  <h5>
- <font color="black"> 5 </font>
-    </h5>
-         <p>
-Laboratory personnel have specific training in the procedures conducted in the laboratory.
-         </p>
-   </div>
-   <div class="caption7">
-  <h5>
- <font color="black"> 6 </font>
-    </h5>
-         <p>
-Supervised by a scientist with proper training.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 7 </font>
-    </h5>
-         <p>
-Contaminated materials are left in open waste receptacles.
-         </p>
-   </div>
-</font>
-</div>
-        </section>
-    <!-- Section #9 -->
-        <section id="maintext2" data-speed="10" data-type="background">
-<br><Br>
   <h5 style="text-align:center;">
   <font color="black">
-BSL 2
+Ligation
 </font>
 </h5>
-<br><br>
+<br>
-<div class="inner2">
- <font color="black">
+<p style="text-align:center">
-   <div class="caption5">
+Quick Ligation Protocol
+</p>
-  <h5>
- <font color="black"> 1 </font>
-    </h5>
-         <p>
-Similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment.
-         </p>
-   </div>
-   <div class="caption5">
+<div class="inner1">
-  <h5>
+<P style="text-align:left;">
+ <font color="black">
+<b>1.</b> Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10μl with dH2O. <br>
+<b>2.</b> Add 10μl of 2X Quick Ligation Reaction Bugger and mix. <br>
- <font color="black"> 2 </font>
+<b>3.</b> Add 1μl of Quick T4 DNA Ligase and mix thoroughly. <br>
-    </h5>
-         <p>
-Includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting such as C. difficile, most Chalmydiae, hepatitis A,B, and C, orthopoxviruses, influenza A, Lyme disease, Salmonella, mumps, measles, scapie, MRSA, and VRSA.
-         </p>
-   </div>
+<b>4.</b> Centrifuge briefly and incubate at room temperature (25°C) for 5mins. <br>
-   <div class="caption5">
+<b>5.</b> Chill on ice, then transform or store at -20°C <br>
-  <h5>
- <font color="black"> 3 </font>
+<b>6.</b> Do not heat inactivate. Heat activation dramatically reduces transformation efficiency. <br>
-    </h5>
+</p>
-         <p>
+</div>
-similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting.
-         </p>
-   </div>
-   <div class="caption3">
+ <h5 style="text-align:center;">
-  <h5>
+  <font color="black">
+Enzyme Digestion
-  <font color="black"> 4 </font>
+</font>
-    </h5>
+</h5>
-         <p>
+<br>
-Laboratory personnel have specific training in handling pathogenic agents,
-         </p>
-   </div>
-   <div class="caption3">
+                <img class="displayed" src="https://static.igem.org/mediawiki/2015/c/cb/Graphics2-20.png" width="1026px">
-  <h5>
+        </section>
- <font color="black"> 5 </font>
-    </h5>
-         <p>
-Must be directed by scientists with advanced training.
-         </p>
-   </div>
-   <div class="caption3">
-  <h5>
- <font color="black"> 6 </font>
+    <!-- Section #11 --!>
-    </h5>
+        <section id="maintext2" data-speed="10" data-type="background">
-         <p>
+                <img class="displayed" src="https://static.igem.org/mediawiki/2015/6/68/Graphics2-21.png" width="1026px">
-Extreme precautions are taken with contaminated sharp items.
+<br>
-         </p>
+                <img class="displayed" src="https://static.igem.org/mediawiki/2015/1/12/Graphics2-22.png" width="1026px">
-   </div>
+        </section>
-   <div class="caption4">
-  <h5>
- <font color="black"> 7 </font>
-    </h5>
-         <p>
-In case that infectious aerosols or splashes may be created, specified procedures should be conducted in biological safety cabinets or other physical containment equipment.
-         </p>
-   </div>
-</font>
+    <!-- Section #12 --!>
-</div>
+        <section id="maintext2" data-speed="10" data-type="background">
+                <img class="displayed" src="https://static.igem.org/mediawiki/2015/1/1a/Graphics2-23.png" width="1026px">
+<br>
+                <img class="displayed" src="https://static.igem.org/mediawiki/2015/c/c7/Graphics2-24.png" width="1026px">
          </section>
+    <!-- Section #13 --!>
+ <h5 style="text-align:center;">
+ <font color="black">
+  Plasmid Construction Protocol
+</font>
+</h5>
+<br>
+     <!-- Section #13 --!>
-     <!-- Section #10 --!>
          <section id="maintext" data-speed="10" data-type="background">
   <h5 style="text-align:center;">
-                <font color="black">
+ <font color="black">
-BSL 3
+Preliminary Work
 </font>
 </h5>
 <br>
-<div class="inner3">
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/5/59/Graphics2-25.png" width="1026px">
-<font color="black">
-   <div class="caption4">
-  <h5>
+<h5 style="text-align:center;">
-  <font color="black"> 1 </font>
+  <font color="black">
-    </h5>
+Material
-         <p>
+</font>
-Indigenous or exotic agents which may cause serious or potentially lethal disease after inhalation.
+</h5>
-         </p>
+<br>
-   </div>
-   <div class="caption4">
+</section>
-  <h5>
- <font color="black"> 2 </font>
-    </h5>
-         <p>
-Includes various bacteria, parasites and viruses that can cause severe to fatal disease in humans but for which  treatments exist, such as Yersinia pestis, SARS coronavirus, Brucella, yellow fever virus.
-         </p>
-   </div>
-   <div class="caption4">
+    <!-- Section #14 --!>
-  <h5>
+        <section id="maintext" data-speed="10" data-type="background">
- <font color="black"> 3 </font>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/5/59/Graphics2-25.png" width="1026px">
-    </h5>
-         <p>
-Laboratory personnel have specific training in handling pathogenic and potentially lethal agents,
-         </p>
-   </div>
-   <div class="caption4">
+ <h5 style="text-align:center;">
-  <h5>
+  <font color="black">
-  <font color="black"> 4 </font>
+Lab Work for Plasmid Construction
-    </h5>
+</font>
-         <p>
+</h5>
-Supervised by competent scientists who are experienced in working with these agents.
+<br>
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 5 </font>
-    </h5>
-         <p>
-All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets,  specially designed hoods, or other physical containment devices.
-         </p>
-   </div>
-   <div class="caption4">
+</section>
-  <h5>
- <font color="black"> 6 </font>
-    </h5>
-         <p>
-The laboratory personals should wear appropriate personal protective clothing and equipment.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 7 </font>
-    </h5>
-         <p>
-An acceptable level of safety for the conduct of routine procedures, may be achieved in a biosafety level 2 facility, providing the filtered exhaust air from the laboratory room is discharged to the outdoors.
-         </p>
-   </div>
-   <div class="caption4">
+    <!-- Section #15 --!>
-  <h5>
+        <section id="maintext" data-speed="10" data-type="background">
- <font color="black"> 8 </font>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/8/85/Graphics-40.png" width="1026px">
-    </h5>
+<br>
-         <p>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/f/fb/Graphics-41.png" width="1026px">
-The ventilation to the laboratory is balanced to provide directional airflow into the room.
+<br>
-         </p>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/2/2b/Graphics-42.png" width="1026px">
-   </div>
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/2/2b/Graphics-43.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/4/40/Graphics-44.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/0/01/Graphics-45.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/5/52/Graphics-46.png" width="1026px">
+<br>
+</section>
-   <div class="caption4">
-  <h5>
- <font color="black"> 9 </font>
-    </h5>
-         <p>
-Access to the laboratory is restricted when work is in progress and the recommended Standard Microbiological  Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed.
-         </p>
-   </div>
-</font>
-</div>
-        </section>
+    <!-- Section #16 --!>
+        <section id="maintext" data-speed="10" data-type="background">
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/0/01/Graphics-47.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/0/03/Graphics-48.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/5/5e/Graphics-50.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/2/2f/Graphics-51.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/1/15/Graphics-52.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/d/df/Graphics-53.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/8/85/Graphics-40.png" width="1026px">
+<br>
+</section>
+    <!-- Section #17 --!>
+        <section id="maintext" data-speed="10" data-type="background">
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/7/7d/Graphics-54.png" width="1026px">
+<br>
+<img class="displayed" src="https://static.igem.org/mediawiki/2015/c/c1/Graphics-55.png" width="1026px">
+<br>
-    <!-- Section #11 --!>
-        <section id="maintext2" data-speed="10" data-type="background">
   <h5 style="text-align:center;">
   <font color="black">
-BSL 4
+Control Experiment
 </font>
 </h5>
-<br><br>
+<br>
-<div class="inner2">
- <font color="black">
+<div class="inner1">
-   <div class="caption4">
+<P style="text-align:center;">First, let's see a video showing our control experiment work:</p><br /><br /></div>
-  <h5>
+<div align="center">
+<video controls>
+<source src="https://static.igem.org/mediawiki/2015/0/05/Experiment_full_version.mp4" type="video/mp4">
+</video>
+</div>
+<br /><br />
- <font color="black"> 1 </font>
+<div class="inner1">
-    </h5>
+<P style="text-align:left;">
-         <p>
+ <font color="black">
-Limited access only for authorized persons can work in the facilities. Not allowed for people who have weak immune system.
+Purpose: investigate the concentration of AHL; <br>
-         </p>
+Find out how much AHL concentration is optimal and how much time it takes for the AHL to express GFP; <br>
-   </div>
+Find out how much time it takes for the test cell to break down AHL <br>
-   <div class="caption4">
+Protocol:<br>
-  <h5>
+<b>1.</b> Incubate bacteria overnight at 37C<br />
+<b>2.</b> Separate out 1ml of test cell and 1ml of reporter cell<br />
+<b>3.</b> Add 3ul of AHL to the test cell<br />
+<b>4.</b> Incubate for 30 minutes<br />
+<b>5.</b> Add the reporter to the test cell<br />
+<b>6.</b> Incubate for 3 hours<br />
+<b>7.</b> Move 200ul of the solution into a well plate<br />
+<b>8.</b> Put the well plate into the spectronometer<br />
+<b>9.</b> View the results<br />
- <font color="black"> 2 </font>
+</font>
-    </h5>
+</p>
-         <p>
+</div>
-When dealing with biological hazards at this level, the use of a positive pressure personnel suit, with a segregated  mandatory.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 3 </font>
-    </h5>
-         <p>
-All works should be supervised by qualified scientists who are trained and experienced in working with these agents.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 4 </font>
-    </h5>
-         <p>
-The entrance and exit of a level 4 bio-lab will contain multiple showers, a vacuum room, an ultraviolet light room, and other safety precautions designated to destroy all traces of the biohazard.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 5 </font>
-    </h5>
-         <p>
-Multiple airlocks are employed and are electronically secured to prevent both doors from opening at the same time.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 6 </font>
-    </h5>
-         <p>
-All air and water service going to and coming from a biosafety level 4 will undergo similar decontamination  procedures to eliminate the possibility of an accidental release.
-         </p>
-   </div>
-   <div class="caption4">
-  <h5>
- <font color="black"> 7 </font>
-    </h5>
-         <p>
-Members of the laboratory staff shall have specific and thorough training in handling extremely hazardous infectious agents, and they shall understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics.
-         </p>
-   </div>
-</font>
-</div>
-        </section>
+</section>
 <script>