Difference between revisions of "Team:Tianjin/Protocol"

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TEAM TIANJIN

Team Tianjin-Attribution

General Protocols

1.Plasmid Extraction

We use the TIANprep Mini Plasmid Kit made by TIANGEN Biotech Co.,Ltd. to extract plasmid. Here is the protocol.
Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the adding volume.
1. Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube. (Please use freshly treated spin column).
2. Harvest 1-5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15-25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.)
3. Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.
Note: No cell clumps should be visible after resuspension ofthe pellet, otherwise incomplete lysis will lower yield and purity. 4. Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times.
Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.
5. Add 350 μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge.
Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3. If there is still white precipitation in the supernatant, please centrifuge again.
6. Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.
7. (Optional, actually we hardly ever use) Wash the Spin Column CP3 by adding 500 μl Buffer PD and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and put Spin Column CP3 back to the collection tube.
This step is recommended to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content.
8. Wash the Spin Column CP3 by adding 600 μl Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube.
9. Repeat Step 8.
10. Centrifuge for an additional 2 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW.
Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP3 lid and stay at room temperature for a while to get rid of residual ethanol.
11. Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50-100 μl Buffer EB to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
Note: If the volume of eluted buffer is less than 50 μl, it may affect recovery efficiency. The pH value of eluted buffer will have some influence in eluting; Buffer EB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 11 to increase plasmid recovery efficiency.

2.DNA Purification

We use the TIANquick Midi Purification Kit made by TIANGEN Biotech Co.,Ltd. to purify the DNA products of PCR and restriction endonuclease cutting. Here is the protocol.
Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume).
1. Column equilibration: add 500 μl Buffer BL to the Spin Column CB2 (put Spin Column CB2 into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 × g). Discard the flow-through, and then place Spin Column CB2 back into the collection tube (please use freshly treated spin column).
2. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix. It is not necessary to remove mineral oil or kerosene.
Note: For example, add 250 μl Buffer PB to 50 μl PCR reaction (not including oil).
3. Transfer the mixture to the Spin Column CB2, incubate at room temperature (15-25°C) for 2 min. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB2 back into the same collection tube.
Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again.
4. Add 600 μl Buffer PW (ensure that ethanol (96-100%) has been added) to the Spin Column CB2 and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and place Spin Column CB2 back in the same collection tube.
Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5 min after adding Buffer PW, and then centrifuge.
5. Repeat step 4.
6. Centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry the membrane.
Note: Residual ethanol from Buffer PW may inhibit subsequent experiment (enzymatic or PCR reactions).
7. Place the Spin Column CB2 in a clean 1.5 ml microcentrifuge tube. Add 30-50 μl Buffer EB to the center of membrane, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
Note: If the volume of eluted buffer is less than 30 μl, it may affect recovery efficiency. The pH value of eluted buffer will have big influence in eluting; distilled water (pH 7.0-8.5, adjusted with NaOH) is suggested to elute plasmid DNA, pH<7.0 will decrease elution efficiency. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 7 to increase plasmid recovery efficiency.

3.Agarose Gel Electrophoresis Products Recycling

We use the TIANgel Midi DNA Purification Kit made by TIANGEN Biotech Co.,Ltd. to recycle the DNA products from the agarose gel. Here is the protocol.
Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume).
1. Column equilibration: add 500 μl Buffer BL to the Spin Column CA2 (put Spin Column CA2 into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put Spin Column CA2 back into the collection tube (please use freshly treated spin column).
2. Cut the DNA fragment from agarose gel with a clean, sharp scalpel. Weigh the gel slice in a clean tube.
3. Add equivalent volume of Buffer PN to the gel (If the gel is 0.1 g, it is defaulted to be 100 μl, then add 100 μl Buffer PN). Incubate at 50°C by inverting up and down the tube until the agarose gel dissolves completely. If the agarose gel does not dissolve completely, incubate for longer period or add additional Buffer PN until all the agarose gel dissolved completely (If the agarose gel is too large, please cut the agarose gel into several pieces in advance).
Note: If DNA fragment is <300 bp, it is recommended to add isopropanol which is 1/2 volume of Buffer PN to the agarose gel sample after the gel completely dissolved. Cooling the solution at room temperature (15-25°C) and then add the solution to Spin Column CA2 since silica membrane of the column adsorbs DNA best at room temperature.
4. When the gel dissolved completely and the solution temperature turns to room temperature (15-25°C), transfer the mixture to the Spin Column CA2 (put Spin Column CA2 into a collection tube). Let the column stand for 2 min at room temperature (15-25°C), then centrifuge for 30-60 s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through; place the Spin Column CA2 back into the collection tube again.
Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again.
5. Wash the Spin Column CA2 with 600 μl Buffer PW (ensure that ethanol (96-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and place the Spin Column CA2 back into the collection tube.
Note: If the purified DNA is used for the salt sensitive experiments, such as direct sequencing and blunt-ended ligation, let the column stand for 2-5 min after adding Buffer PW, and then centrifuge.
6. Repeat Step 5.
7. Place the Spin Column CA2 back to the collection tube and centrifuge at 12,000 rpm (~13,400 × g) for 2 min to remove residual wash buffer. Discard the flow-through, and place column with the cap open for several minutes to air dry the membrane.
Note: Residual ethanol from Buffer PW will influence the subsequent enzymatic reaction (enzyme digestion, PCR etc).
8. Transfer the Spin Column CA2 to a clean 1.5 ml microcentrifuge tube. Add appropriate volume of Buffer EB to the center of the membrane, incubate at room temperature (15-25°C) for 2 min, then centrifuge at 12,000 rpm (~13,400 × g) for 2 min.
Note: The elution volume should not be less than 30 μl since smaller volume will affect recovery efficiency. The pH value of eluted buffer will affect eluting. If purified DNA is used for sequencing, it is recommended to choose ddH₂O (pH 7.0-8.5) to elute DNA, pH<7.0 will decrease the elution efficiency. Obtained DNA should be stored at -20°C to prevent degradation. Buffer (10 mM Tris-Cl, pH 8.0) could also be used for DNA elution. For higher yield, pipette the eluate to the center of the membrane again, incubate 2 min and centrifuge at 12,000 rpm (~13,400 × g) for 2 min.

4.Agarose Gel Electrophoresis

1. Preparation of TAE buffer: Add 242g Tris, 37.2g Na2EDTA·2H2O, 800mL ddH₂O, after all the solute dissolve, add 57.1mL acetic acid and add ddH₂O to make the total volume 1L.
2. Preparation of sample: Add DNA loading buffer to the DNA solution according to the dilution ratio of particular buffer.
3. Preparation of agarose gel: Add agarose powder 10g/L, 1×TAE buffer 100mL (variable, according to need), heat the mixture by microwave oven until the agarose was dissolved. After the solution cool down to touchable temperature, add 50-100μL/L Goldenview Nucleic acid dye to the solution. Then pour the solution to the gel mould with gel comb inserted and wait for its concretion.
4. Add samples to the gel pore: After the formation of gel, pull out the gel comb and take the gel out of the mould. Immerge the gel with 1×TAE buffer in the electrophoresis chamber. Using pipette to add marker and samples to different pore. (The content of pore depends on the gel comb, there are 3 kinds of volume, 10μL, 20μL, and 50μL) Do not stick the bottom and side of gel pore to prevent the leakage.
5. Turn on the electrical source to start the electrophoresis, the voltage is set at 150-160V and the electrophoresis time is set at 8-12min.
6. After the electrophoresis process end, the gel is observed under blue light or ultraviolet.

5.Restriction Endonuclease Digestion

Prepare the system:

Total volume: 50μL

Restriction endonuclease: 2μL respectively

10×Cut Smart Buffer: 5μL

DNA to be cut: 30μL

ddH₂O: 13μL

Reaction time: 2h

Reaction temperature: 37℃

6.DNA Fragments Ligation

Prepare the system:

Total volume: 20μL

T4 DNA ligase: 1μL

5×Ligase Buffer: 4μL

DNA to be linked: (c1V1/L1): (c2V2/L2)=5:1. (c1: Concentration of cut DNA fragments; c2: Concentration of cut plasmid; V1: Volume of cut DNA fragments; V2: Volume of cut plasmid; L1: Length of cut DNA fragments; L2: Length of cut plasmid)

ddH₂O: add to make the total volume 20μL

Reaction time: 2h

Reaction temperature: 22℃

7.PCR

Prepare the system:

Total volume: 50μL

Q5 DNA Polymerase: 0.5μL

5×Q5 DNA Polymerase Buffer: 10μL

Template: 1μL

dNTP: 1μL

Primer: Sense Primer and Anti-sense Primer, respectively 2.5μL

ddH₂O: 32.5μL

Cycles: 25-35

Pre-denaturation: 98℃,30s

Denaturation: 98℃,5-10s

Annealing: Depend on the primers, generally 45-65℃,10-30s

Extension: 72℃,20-30s/kb

Fully extension: 72℃,2min

Product Storage: 4℃

Note: Different DNA polymerase has different protocol, this is only the case of Q5 DNA polymerase.

8.Colony PCR

Prepare the system:

Total volume: 20μL

Colony PCR System: 2×Taq Master Mix (Dye added) 10μL

ddH₂O: 10μL

select single colony with stick and immerse the stick into the system for 1 min. Preparing the bacterial liquid and then add 1μL of it is also feasible.

Cycles: 25-35

Pre-denaturation: 94℃,5min

Denaturation: 94℃,30s

Annealing: Depend on the primers, generally 45-65℃,20-60s

Extension: 72℃, depend on the length of amplified fragment, generally 20-60s

Fully extension: 72℃,7min

Product Storage: 4℃

Note: Preparation of bacteria liquid Prepare the system:

Total volume: 50μL

ddH₂O: 50μL

select single colony with stick and immerse the stick into the system for 1 min

Cycles: 3-4

Pre-denaturation: 99℃,4min

Renaturation: 4℃,4min

Product Storage: 4℃

9.TransT1 Transformation

1. Mix 50μL competent E.coli cell with 10μL plasmids, and place them in ice bath for 30 min.
2. Heat shock at 42℃ for 30 s.
3. Put the system back on ice for 2 min.
4. Add 500 μl of LB without antibiotics and incubate at 37 ℃ for 30-60 min with the shaking speed of 200rpm.
5. Centrifuge the culture medium at 4000 rpm for 3min.
6. Discard the supernatant and resuspend the sediment, use this resuspending liquid to spread the plate with antibiotics.

Protocols for Microbial Consortia

1.Culture Medium of E.coli-Sc Microbial consortia

13.3 g/L KH2PO4

4 g/L (NH4)2HPO4

1.7 g/L citric acid

0.0084 g/L EDTA

0.0025 g/L CoCl2

0.015 g/L MnCl2

0.0015 g/L CuCl2

0.003 g/L H3BO3

0.0025 g/L Na2MoO4

0.008 g/L Zn(CH3COO)2)

0.06 g/L Fe(III) citrate

0.0045 g/L thiamine

1.3 g/L MgSO4

40 g/L glucose

5 g/L yeast extract

20 g/L xylose

2.16s-DNA

Prepare the system:

Total volume: 20μL

Colony PCR System: 2×Taq Master Mix (Dye added) 10μL

ddH₂O: 8.2μL

primer: 0.4μL each

bacteria liquid:1μL

The primer could copy the special 16s-DNA from this kind of bacteria.

The bacteria liquid is pretreatment.

Cycles: 25-35

Pre-denaturation: 94℃,2min

Denaturation: 94℃,30s

Annealing: Depend on the primers, generally 45-65℃,20-60s

Extension: 72℃, depend on the length of amplified fragment, generally 20-60s

Fully extension: 72℃,4min

Product Storage: 4℃

Protocols for Cell-Free Protein Synthesis

1.Cell-free protein synthesis system(50 µL)

ddH₂O:7.9µL

Feeding buffer : 25µL

Mg2+ solution : 1.1µL

Gene( plasmid as template) : 1µL

Lysate : 15µL

2.Preparation of pNPA degradation reaction system

Substrate: 0.2mM pNPA water solution (0.00362g pNPA dissolving in ddH₂O)
Reaction system: 10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate. (pH=7)
After static reaction at 39℃ for 8 hours, we detected the characteristic adsorption peak of the product ,pNP, which has no other characteristic adsorption peak except in 400nm.

2.Preparation of PET degradation reaction system

Reaction system: PET was put into 20 times diluted Enzyme solution(unpurified).(pH=7)
After static reaction at 39℃ for 2 days, we detected the characteristic adsorption peak of the product ,MHET, which has no other characteristic adsorption peak except in 260nm. And detect it every other day in the future 3-4 days.

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Notice: This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release. — 2016 iGEM Team Tianjin

@@ Line 166: / Line 166: @@
 Note: Residual ethanol from Buffer <b>PW</b> will influence the subsequent enzymatic reaction (enzyme digestion, PCR etc). <br/>
 . Transfer the Spin Column<b> CA2</b> to a clean <b>1.5 ml</b> microcentrifuge tube. Add appropriate volume of Buffer<b> EB </b>to the center of the membrane, incubate at room temperature (<b>15-25°C</b>) for <b>2 min</b>, then centrifuge at <b>12,000 rpm (~13,400 × g)</b> for <b>2 min</b>. <br/>
-Note: The elution volume should not be less than <b>30 μl</b> since smaller volume will affect recovery efficiency. The pH value of eluted buffer will affect eluting. If purified DNA is used for sequencing, it is recommended to choose ddH2O (<b>pH 7.0-8.5</b>) to elute DNA,    <b>pH<7.0</b> will decrease the elution efficiency. Obtained DNA should be stored at<b> -20°C</b> to prevent degradation. Buffer (<b>10 mM Tris-Cl, pH 8.0</b>) could also be used for DNA elution. For higher yield, pipette the eluate to the center of the membrane again, incubate <b>2 min</b> and centrifuge at <b>12,000 rpm (~13,400 × g)</b> for <b>2 min</b>.
+Note: The elution volume should not be less than <b>30 μl</b> since smaller volume will affect recovery efficiency. The pH value of eluted buffer will affect eluting. If purified DNA is used for sequencing, it is recommended to choose ddH<sub>2</sub>O (<b>pH 7.0-8.5</b>) to elute DNA,    <b>pH<7.0</b> will decrease the elution efficiency. Obtained DNA should be stored at<b> -20°C</b> to prevent degradation. Buffer (<b>10 mM Tris-Cl, pH 8.0</b>) could also be used for DNA elution. For higher yield, pipette the eluate to the center of the membrane again, incubate <b>2 min</b> and centrifuge at <b>12,000 rpm (~13,400 × g)</b> for <b>2 min</b>.
 </div>
@@ Line 193: / Line 193: @@
 <div class="note-content4">
-. Preparation of <b>TAE buffer</b>: Add <b>242g Tris, 37.2g Na2EDTA·2H2O, 800mL ddH2O</b>, after all the solute dissolve, add <b>57.1mL acetic acid </b>and add <b>ddH2O</b> to make the total volume <b>1L</b>.<br/>
+. Preparation of <b>TAE buffer</b>: Add <b>242g Tris, 37.2g Na2EDTA·2H2O, 800mL ddH<sub>2</sub>O</b>, after all the solute dissolve, add <b>57.1mL acetic acid </b>and add <b>ddH<sub>2</sub>O</b> to make the total volume <b>1L</b>.<br/>
 . Preparation of sample: Add <b>DNA loading buffer</b> to the DNA solution according to the dilution ratio of particular buffer.<br/>
 . Preparation of agarose gel: Add <b>agarose powder 10g/L, 1×TAE buffer 100mL </b>(variable, according to need), heat the mixture by microwave oven until the agarose was dissolved. After the solution cool down to touchable temperature, add <b>50-100μL/L Goldenview Nucleic acid dye</b> to the solution. Then pour the solution to the gel mould with gel comb inserted and wait for its concretion.<br/>
@@ Line 227: / Line 227: @@
 <li>10×Cut Smart Buffer:<b> 5μL</b></li>
 <li>DNA to be cut: <b>30μL</b></li>
-<li>ddH2O:<b> 13μL</b></li>
+<li>ddH<sub>2</sub>O:<b> 13μL</b></li>
 <br/>
 <li>Reaction time: <b>2h</b></li>
@@ Line 265: / Line 265: @@
 <li>5×Ligase Buffer:<b> 4μL</b></li>
 <li>DNA to be linked: <b>(c1V1/L1): (c2V2/L2)=5:1</b>. (c1: Concentration of cut DNA fragments; c2: Concentration of cut plasmid; V1: Volume of cut DNA fragments; V2: Volume of cut plasmid; L1: Length of cut DNA fragments; L2: Length of cut plasmid)</li>
-<li>ddH2O: add to make the total volume <b>20μL</b></li>
+<li>ddH<sub>2</sub>O: add to make the total volume <b>20μL</b></li>
 <br/>
 <li>Reaction time:<b> 2h</b></li>
@@ Line 297: / Line 297: @@
 <li>dNTP:<b> 1μL</b></li>
 <li>Primer: Sense Primer and Anti-sense Primer, respectively <b>2.5μL</b></li>
-<li>ddH2O: <b>32.5μL</b></li>
+<li>ddH<sub>2</sub>O: <b>32.5μL</b></li>
 <br/>
 <li>Cycles: <b>25-35</b></li>
@@ Line 336: / Line 336: @@
 <li>Total volume: <b>20μL</b></li>
 <li>Colony PCR System: 2×Taq Master Mix (Dye added) <b>10μL</b></li>
-<li>ddH2O: <b>10μL</b></li>
+<li>ddH<sub>2</sub>O: <b>10μL</b></li>
 <li>select single colony with stick and immerse the stick into the system for <b>1 min</b>. Preparing the bacterial liquid and then add 1μL of it is also feasible.
 </li><br/>
@@ Line 350: / Line 350: @@
 Prepare the system:
 <li>Total volume: <b>50μL</b></li>
-<li>ddH2O: <b>50μL</b></li>
+<li>ddH<sub>2</sub>O: <b>50μL</b></li>
 <li>select single colony with stick and immerse the stick into the system for <b>1 min</b>
 </li><br/>
@@ Line 439: / Line 439: @@
 <li>Total volume: <b>20μL</b></li>
 <li>Colony PCR System: 2×Taq Master Mix (Dye added) <b>10μL</b></li>
-<li>ddH2O: <b>8.2μL</b></li>
+<li>ddH<sub>2</sub>O: <b>8.2μL</b></li>
 <li>primer: <b>0.4μL </b> each</li>
 <li>bacteria liquid:<b>1μL</b></li>
@@ Line 472: / Line 472: @@
 <div class="note-content12">
-<li>ddH2O:<b>7.9µL</b></li>
+<li>ddH<sub>2</sub>O:<b>7.9µL</b></li>
 <li>Feeding buffer :   <b>25µL</b></li>
 <li>Mg2+ solution  :  <b>1.1µL</b></li>
@@ Line 496: / Line 496: @@
 <div class="note-content13">
-Substrate: <b>0.2mM pNPA </b>water solution (<b>0.00362g pNPA </b>dissolving in ddH2O)<br/>
+Substrate: <b>0.2mM pNPA </b>water solution (<b>0.00362g pNPA </b>dissolving in ddH<sub>2</sub>O)<br/>
 Reaction system: <b>10 times</b> diluted Enzyme solution(unpurified) mixed with the equal volume substrate. (<b>pH=7</b>)<br/>
 After static reaction at <b>39℃ </b>for <b>8 hours</b>, we detected the characteristic adsorption peak of the product ,pNP, which has no other characteristic adsorption peak except in <b>400nm</b>.