Team:Tianjin/Protocol

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Protocol

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.
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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.
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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 CA2into 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 CA2into 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.
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Week4(9/14/2016-9/20/2016)

  • We started to construct another regulation way, the E.coli lysis regulation pathway. We first used colony PCR to obtain the ddpX gene from the E.coli genome and recycled the ddpX from the agarose gel.
  • We found that there was no enzyme cleavage site between the CpxR promoter and RFP gene in the part we use. We had to design the primers and amplified the CpxR promoter by PCR.
  • We used PCR to amplify the CpxR promoter. Then we recycled it from agarose gel.
  • We cut the CpxR promoter with enzymes Xba1 and BamH1, ddpX gene with enzymes BamH1 and EcoR1, first batch of pET21a with Xba1 and EcoR1, second batch of pET21a with BamH1 and EcoR1.
  • Then we linked these fragment in the following two ways:
    1. pET21a-CpxR-ddpX.
    2. pET21a-ddpX.
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    Week5(9/21/2016-9/27/2016)

  • We used PCR to amplify the whole fragments in pET21a (from CpxR to T7 terminator). However, the band in the agarose gel was disperse so that we were unable to recycle it.
  • We used colony PCR to verify if the pET21a had been correctly constructed, the result is yes.
  • We changed the DNA polymerase and annealing temperature several times and redid the PCR, however, the disperse band were always existed.
  • We cultured the E.coli transformed into the pET21a-ddpX fragment and detect the OD600 in order to verify the lysis effection of ddpX.
  • Considering the pYES2 is multicopy plasmid so that the copy number would affect the RFP expression level, we decided to change the pYES2 to single-copy plasmid pRS416. Since the pRS416 does not have terminator in its backbone, we used PCR to amplify the CYC1 terminator from plasmid pYES2.
  • We cut the pYES2 with enzyme Hind3 and EcoR1, CYC1 with EcoR1 and Sal1, pRS416 with Hind3 and Sal1. Then we linked the three part together.
  • We transformed the three plasmids into Saccharomyces cerevisiae together.
  • The new primers using to amplify the CpxR-ddpX-T7 terminator fragment arrived and we redid the PCR. However, the disperse band was still existed.
  • The transformation of Saccharomyces cerevisiae turned out to be a failure because no colony was found on the Sc-Ura-Leu-His plate.
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    Week6(9/28/2016-10/2/2016)

  • We redid the inclusion body reporting experiment, and this time we directly observed the color of bacterial after centrifugation (12000rpm, 1min). The group with PETase gene and CpxR-RFP fragment showed the deepest red.
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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

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