Team:BIT-China/Protocol
3
Basic Protocol
Protocol for PCR
A: Protocol for PCR with Pfu DNA polymerase [Expand]
1. Prepare the reaction mix in PCR tube:
| Template DNA | 1µl |
| Forward primer | 2µl |
| Reverse primer | 2µl |
| dNTP | 2µl |
| 10X Pfu buffer | 2.5µl |
| Pfu DNA polymerase | 0.5µl |
| ddH20 | 15µl |
| Total volume | 25µl |
When we ligate two fragments by OE-PCR, we usually add two kinds of fragment DNA 1 µl respectively and add ddH20 up to 25µl.
2. Set the program of thermal cycler:
| 1X | 95℃ | 10min |
| 30X | 95℃ | 30s |
| 30X | 55℃ | 30s |
| 30X | 72℃ | 800bp/min |
| 1X | 72℃ | 10min |
| 1X | 16℃ | ∞ |
B: Protocol for PCR with PrimeStar DNA polymerase [Expand]
1. Prepare the reaction mix in PCR tube:
| Template DNA | 1.5µl |
| Forward primer | 1.5µl |
| Reverse primer | 1.5µl |
| dNTP | 2µl |
| 2X CG buffer | 12.5µl |
| PrimeStar DNA polymerase | 0.25µl |
| ddH20 | 5.75µl |
| Total volume | 25µl |
When we ligate two fragments by OE-PCR, we usually add two kinds of fragment DNA
1.5 µl respectively and add ddH20 up to 25µl.
2. Set the program of thermal cycler:
| 1X | 98℃ | 1min |
| 30X | 98℃ | 30s |
| 30X | 55℃ | 7s |
| 30X | 72℃ | 1000bp/min |
| 1X | 72℃ | 5min |
| 1X | 16℃ | ∞ |
C: Protocol for PCR With FastPfu Fly DNA polymerase [Expand]
1. Prepare the reaction mix in PCR tube:
| Template DNA | 1µl |
| Forward primer | 2µl |
| Reverse primer | 2µl |
| dNTP | 2µl |
| 5X FastPfu Fly buffer | 5µl |
| FastPfu Fly DNA polymerase | 0.5µl |
| ddH20 | 12.5µl |
| Total volume | 25µl |
When we ligate two fragments by OE-PCR, we usually add two kinds of fragment DNA 1 µl respectively and add ddH20 up to 25µl.
2. Set the program of thermal cycler:
| 1X | 94℃ | 5min |
| 30X | 94℃ | 30s |
| 30X | 55℃ | 30s |
| 30X | 72℃ | 1500bp/min |
| 1X | 72℃ | 10min |
| 1X | 16℃ | ∞ |
Protocol for Agarose Gel Electrophoresis [Expand]
1. Measure TAE buffer.
2. Weigh agarose powder and add agarose powder to the buffer.
3. Melt the mixture in a microwave until the solution becomes clear.
4. Let the solution cool to about 40-50℃ and add nucleic acid dye.
5. Pour the solution into the gel tray with corresponding comb.
6. Let the gel cool until it becomes solid.
7. Carefully pull out the comb and place the gel in the electrophoresis chamber.
8. Add enough TAE Buffer.
9. Pipette DNA samples mixed with appropriate amount of loading buffer and dye (Gene Finder) into wells on the gel;
10. Run the gel at 60V for 20 minutes.
2. Weigh agarose powder and add agarose powder to the buffer.
3. Melt the mixture in a microwave until the solution becomes clear.
4. Let the solution cool to about 40-50℃ and add nucleic acid dye.
5. Pour the solution into the gel tray with corresponding comb.
6. Let the gel cool until it becomes solid.
7. Carefully pull out the comb and place the gel in the electrophoresis chamber.
8. Add enough TAE Buffer.
9. Pipette DNA samples mixed with appropriate amount of loading buffer and dye (Gene Finder) into wells on the gel;
10. Run the gel at 60V for 20 minutes.
Protocol for digestion [Expand]
1. Prepare reaction mix according to the following table
| Enzyme digestion system (20μL) | |
| Fragment or plasmid need to digest | 16μL |
| corresponding enzymes of upstream restriction Enzyme cutting site | 1μL |
| corresponding enzymes of downstream restriction Enzyme cutting site | 1μL |
| Enzyme buffer (Buffer) | 2μL |
2. incubate at recommended temperature (37℃) for at least 1 hour.
3. Purify the digestion product by inactivating at 80 ° C.
3. Purify the digestion product by inactivating at 80 ° C.
Protocol for ligation [Expand]
1. Prepare reaction mix according to the following table
| T4 DNA Ligase system (10μL) | |
| digested fragment/vector | 8.5μL |
| T4 DNA ligase | 0.5μL |
| 10x T4 DNA ligase buffer | 1μL |
2. Ligate the mix overnight at 16℃.
Note: when 3A assembly is performed, we always adjust the dose of each fragment according to their concentration to make sure the amounts of them are same to each other. Vector can be added less than fragment but no less than 1.5μL.
Protocol for transformation（chemical）[Expand]
1. Take the competent cells (BMTOP10) out of the Ultra-low temperature freezer.
2. Put the competent cells in the ice for 5 minutes.
3. Add 10ul of the ligation product to the competent cells and put them in the ice for 30 minutes.
4. Next put the competent cells in water about 42℃ for 90 seconds. Then put the competent cells in ice for 5 minutes immediately.
5. Add 600ul LB-media to the competent cells and put them into the Incubator shaker for 1 hour in 37℃.
6. Take 100ul of the competent cells into the LB plate containing corresponding antibiotics and spread them on the plate evenly.
7. Put the LB plate to the Incubator for 18 hours.
2. Put the competent cells in the ice for 5 minutes.
3. Add 10ul of the ligation product to the competent cells and put them in the ice for 30 minutes.
4. Next put the competent cells in water about 42℃ for 90 seconds. Then put the competent cells in ice for 5 minutes immediately.
5. Add 600ul LB-media to the competent cells and put them into the Incubator shaker for 1 hour in 37℃.
6. Take 100ul of the competent cells into the LB plate containing corresponding antibiotics and spread them on the plate evenly.
7. Put the LB plate to the Incubator for 18 hours.
Protocol for Gel Purification [Expand]
Protocol for Gel Purification is based on the introduction of Tiangen Gel Extraction Kit.
Protocol for mini-prep [Expand]
Protocol for mini-prep is based on the introduction of Tiangen Mini-prep Kit
Special Protocol
Protocol for Electroporational transformation [Expand]
1. Inoculate 50μl fresh overnight E.coli culture into 50ml LB culture medium with antibiotics.
2. Put the cells at 37°C Incubator shaker at 170rpm until OD600 reaches to 0.5-0.6.
3. Chill the cell and ddH2O in ice for 30 minutes. For all subsequent steps, keep the cells as close to 0 °C as possible and chill all containers in the ice before adding cells.
4. Transfer the cells to an aseptic and cold 50ml centrifuge tube and centrifuge at 5000rpm for 5 minutes at 4°C.
5. Pour off and discard the supernatant carefully.
6. Carefully pour 50ml ice-cold ddH2O into Centrifuge tube at 5000rpm for 5 minutes at 4 °C. Pour off and discard the supernatant carefully.
7. Repeat the 6 step for two more times.
8. Suspend the bacteria in 300μl ddH2O. Put the bacteria divided into three EP tubes.
9. Put Gene Pulser cuvette on ice for 10 minutes. Add the competent cell in the Gene Pulser cuvette then add 4μl DNA, put on ice for several minutes.
10. Place the cuvette in the ShockPod. Electric shock once.
11. Remove the cuvette from the chamber and immediately add 600μl SOB medium to the cuvette. Mix it carefully.
12. Transfer it into EP tube and incubate at 37°C, 200rpm for 1 hour.
13. Spread them evenly on LB plate with corresponding antibiotic.
14. Put the LB plate to the Incubator for 18 hours.
2. Put the cells at 37°C Incubator shaker at 170rpm until OD600 reaches to 0.5-0.6.
3. Chill the cell and ddH2O in ice for 30 minutes. For all subsequent steps, keep the cells as close to 0 °C as possible and chill all containers in the ice before adding cells.
4. Transfer the cells to an aseptic and cold 50ml centrifuge tube and centrifuge at 5000rpm for 5 minutes at 4°C.
5. Pour off and discard the supernatant carefully.
6. Carefully pour 50ml ice-cold ddH2O into Centrifuge tube at 5000rpm for 5 minutes at 4 °C. Pour off and discard the supernatant carefully.
7. Repeat the 6 step for two more times.
8. Suspend the bacteria in 300μl ddH2O. Put the bacteria divided into three EP tubes.
9. Put Gene Pulser cuvette on ice for 10 minutes. Add the competent cell in the Gene Pulser cuvette then add 4μl DNA, put on ice for several minutes.
10. Place the cuvette in the ShockPod. Electric shock once.
11. Remove the cuvette from the chamber and immediately add 600μl SOB medium to the cuvette. Mix it carefully.
12. Transfer it into EP tube and incubate at 37°C, 200rpm for 1 hour.
13. Spread them evenly on LB plate with corresponding antibiotic.
14. Put the LB plate to the Incubator for 18 hours.
Protocol for colony PCR [Expand]
1. Prepare the reaction mix in PCR tube:
| ddH20 | 6.5 µl |
| Forward primer | 0.5µl |
| Reverse primer | 0.5µl |
| 2×Taq PCR Master Mix | 7.5µl |
| Total volume | 15µl |
3. Select positive clones and put them in the PCR tubes.
4. Make sure the bacteria are in the cPCR tubes.
5. Set the program of thermal cycler:
4. Make sure the bacteria are in the cPCR tubes.
5. Set the program of thermal cycler:
| 1X | 94℃ | 10min |
| 25X | 94℃ | 30s |
| 25X | 55℃ | 30s |
| 25X | 72℃ | 1000bp/min |
| 1X | 72℃ | 10min |
| 1X | 16℃ | ∞ |
Protocol for fluorescence measurement [Expand]
1. Inoculate the bacteria in LB medium overnight for activation.
2. Transfer 2ml of the activated bacteria into new LB medium.
3. When the OD600 of culture medium reaches to 0.6, induce the bacteria with inducer.
4. Measure the OD600 and fluorescence intensity once an hour.
5. Measure the OD by ultraviolet spectrophotometer. And measure the fluorescence strength by microplate reader.
2. Transfer 2ml of the activated bacteria into new LB medium.
3. When the OD600 of culture medium reaches to 0.6, induce the bacteria with inducer.
4. Measure the OD600 and fluorescence intensity once an hour.
5. Measure the OD by ultraviolet spectrophotometer. And measure the fluorescence strength by microplate reader.
Protocol for growth curve measurement [Expand]
1. Inoculate the bacteria in LB medium overnight for activation.
2. Transfer 2ml of the activated bacteria into new LB medium.
3. When the OD600 of culture medium reaches to 0.6, induce the bacteria with inducer.
4. Measure the OD600 once an hour.
5. Measure the OD by ultraviolet spectrophotometer.
2. Transfer 2ml of the activated bacteria into new LB medium.
3. When the OD600 of culture medium reaches to 0.6, induce the bacteria with inducer.
4. Measure the OD600 once an hour.
5. Measure the OD by ultraviolet spectrophotometer.
Protocol for SDS PAGE [Expand]
1. Make the separating gel and wait 40 minutes until the gel becomes solid.
13％separating gel
| ddH2O | 2.97ml |
| 30% ACRYLAMIDE | 4.33ml |
| 1.5M Tris-HCl（pH8.8） | 2.5ml |
| 10% SDS | 0.1ml |
| 10% ammonium persulfate | 0.1ml |
| TEMED | 0.004ml |
(The proportions of the components are determined by the specific circumstances)
2. Make the stacking gel and wait 40 minutes until the gel becomes solid.
4％stacking gel
| ddH2O | 3.56ml |
| 30% ACRYLAMIDE | 0.67ml |
| 1.5M Tris-HCl（pH6.8） | 0.63ml |
| 10% SDS | 0.05ml |
| 10% ammonium persulfate | 0.05ml |
| TEMED | 0.005ml |
(The proportions of the components are determined by the specific circumstances)
3. Add sample preparation buffer (1: 1 by volume) to the sample and incubate the mixture for 3 minutes at 100 ° C in a boiling water bath. Note: The protein standard sample (Mark) should be treated with boiling water samples at the same time, whether it needs to add the sample buffer should be based on the specific case of protein standards.
4. Place the gel in the electrophoresis chamber.
5. Add enough Tris-glycine electrophores buffer.
6. Pipette samples and Mark into wells on the gel.
7. Run the gel at 60V until the dye entered the separating gel.
8. Run the gel at 120V until the bromophenol blue reaches the bottom of the separation gel.
9. Take the gel out and dye it about 60 minutes with coomassie brilliant blue.
10. Wash twice with deionized water to terminate staining.
11. Add the destaining solution until the gel plate background faded so that protein bands become clearly.
4. Place the gel in the electrophoresis chamber.
5. Add enough Tris-glycine electrophores buffer.
6. Pipette samples and Mark into wells on the gel.
7. Run the gel at 60V until the dye entered the separating gel.
8. Run the gel at 120V until the bromophenol blue reaches the bottom of the separation gel.
9. Take the gel out and dye it about 60 minutes with coomassie brilliant blue.
10. Wash twice with deionized water to terminate staining.
11. Add the destaining solution until the gel plate background faded so that protein bands become clearly.
Protocol for random mutation [Expand]
1. Random primer sequences were synthesized by GENEWIZ. Named SJTB. With this line as the PCR template. (Ptet-B0032-RFP-B0015)
2. For amplification of circuits containing mutated promoters at 67℃with upstream primers SJTB.
2. For amplification of circuits containing mutated promoters at 67℃with upstream primers SJTB.
| ddH2O | 11.5ul |
| Buffer | 5ul |
| dNTP | 3ul |
| Template | 1ul |
| Upstream primer | 2ul |
| Downstream primer | 2ul |
| Enzyme(Fast Pfu Fly) | 0.5ul |
Target Fragment observed through Gel electrophoresis.
3. Purification with kit from TIANGEN Biotech.
4. Restriction digestion of PCR product and the vector with EcoRI and PstI at 37℃ for 2h20min.
5. Purification of digestion product by running agarose gel electrophoresis. Extract the right band by TIANgel Midi Purification Kit.
6. Ligate the PCR product with vector pSB1C3 by T4 DNA ligase.
6. Transform the ligation product into competent cell TOP 10 and incubate it in LB solid culture plates added Chloramphenicol.
7. Use Colony PCR to select the positive clones.
4. Restriction digestion of PCR product and the vector with EcoRI and PstI at 37℃ for 2h20min.
5. Purification of digestion product by running agarose gel electrophoresis. Extract the right band by TIANgel Midi Purification Kit.
6. Ligate the PCR product with vector pSB1C3 by T4 DNA ligase.
6. Transform the ligation product into competent cell TOP 10 and incubate it in LB solid culture plates added Chloramphenicol.
7. Use Colony PCR to select the positive clones.
| Upstream primer | 0.5ul |
| Downstream primer | 0.5ul |
| MIX | 7.5ul |
| ddH2O | 6.5ul |
Target Fragment observed through Gel electrophoresis.
9. Incubate the positive bacteria in LB medium with chloramphenicol for 18 hours.
10. Test the promoter strength through observing the RFP strength.
11. Screen out the intensity we desire and sequence it to confirm the mutated promoter sequence
10. Test the promoter strength through observing the RFP strength.
11. Screen out the intensity we desire and sequence it to confirm the mutated promoter sequence
Protocol for Gibson Assembly
Reaction Stock Preparation [Expand]
1. Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:
• 3 ml of 1 M Tris-HCl pH 7.5
• 150 μl of 2 M MgCl2
• 60 μl of 100 mM dGTP
• 60 μl of 100 mM dATP
• 60 μl of 100 mM dTTP
• 60 μl of 100 mM dCTP
• 300 μl of 1 M DTT
• 1.5 g PEG-8000
• 300 μl of 100 mM NAD
• Add water to 6 ml
• Aliquot 100 μl and store at -20℃
• 150 μl of 2 M MgCl2
• 60 μl of 100 mM dGTP
• 60 μl of 100 mM dATP
• 60 μl of 100 mM dTTP
• 60 μl of 100 mM dCTP
• 300 μl of 1 M DTT
• 1.5 g PEG-8000
• 300 μl of 100 mM NAD
• Add water to 6 ml
• Aliquot 100 μl and store at -20℃
2. Prepare master mixture. Combine the following:
• 320 μl 5X ISO buffer
• 0.64 μl of 10 U/μl T5 exonuclease
• 20 μl of 2 U/μl Phusion polymerase
• 160 μl of 40 U/μl Taq ligase
• Add water to 1.2 ml
• Aliquot 15 μl and store at -20℃
• 0.64 μl of 10 U/μl T5 exonuclease
• 20 μl of 2 U/μl Phusion polymerase
• 160 μl of 40 U/μl Taq ligase
• Add water to 1.2 ml
• Aliquot 15 μl and store at -20℃
Reaction Procedure [Expand]
1. Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
2. Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Total DNA to be assembled should range between 20-200 ng. For large DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
3. Thoroughly mix gently.
4. Incubate at 50℃ for 60 min.
5. Transfer 5 μl to competent cell
6.Similar with common heat shock transformation
2. Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Total DNA to be assembled should range between 20-200 ng. For large DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
3. Thoroughly mix gently.
4. Incubate at 50℃ for 60 min.
5. Transfer 5 μl to competent cell
6.Similar with common heat shock transformation
