Team:Copenhagen/Protocols
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PCR reaction for amplification of backbone pSB1C3
To each PCR tube the following is added:
2,5 μl of each primer
4 μl template DNA
41 μl MasterMix
The PCR programme is as follows:
1. Int. Denaturation 〖95〗^∘ 120sec
2. Denaturation 〖95〗^∘ 30sec
3. Tm 〖55〗^∘ 30sec
4. Elongation 〖68〗^∘ 1.25 min
5. Final Extension 〖68〗^∘ 10min
6. Hold 〖10〗^∘
** Step 2-4 is repeated 32 times
Colony PCR
20 μl H2O is added to each PCR tube. Colonies are chosen from the plates and resuspended in the PCR tubes. This serves as the template solution.
To each PCR tube the following is added:
0,5 μl of each primer
3 μl template DNA
5 μl MangoMixTM (5 mM) (Bioline)
MangoMixTM
MangoTaq DNA polymerase
Orange reference dye
MgCl2
dNTP
Protocol for PCR reactions for individual genes
The following protocol is used to amplify the individual genes.
To each PCR tube the following is added:
2,5 μl of each primer
0,5 μl template DNA
44,5 μl MasterMix
MasterMix:
10 mM X7 Buffer
10 mM DNTPs
2 mM X7 DNA polymerase (produced in house)
The PCR programme used:
1. Int. Denaturation 〖98〗^∘ 30sec
2. Denaturation 〖98〗^∘ 10sec
3. Tm 〖60〗^∘ 20sec
4. Elongation 〖72〗^∘ *sec
5. Final Extension 〖72〗^∘ 7min
6. Hold 〖10〗^∘
*= elongation time for X7 DNA polymerase should be calculated so it is compatible with the size of the template. X7 polymerase writes 1kb/30-45sec.
** Step 2-4 is repeated 32 times
USER Cloning
Procedure:
- The PCR product must be purified from a gel before (GenEluteTM HP plasmid MiniPrep kit (Sigma-Aldrich)) used in USER cloning.
- The PCR products is added to Eppendorf tubes to give a total volume of 8 µl in each tube
(Thus, in case of two PCR products, add 4 µl and 4 µl or 3 µl and 5 µl etc.)
- The USER mix components are mixed: (in each tube)
| USER Mix |
1x USER Mix |
| NEBuffer 4 (10x diluted) |
0.5 µL |
| BSA |
0.5 µL |
| Dpn1 |
1 µL |
- The USER mix is transferred to each Eppendorf tube and incubated for 2 hours at 37°C.
- 1 µL USER enzyme is added pr. tube and the mixture is incubated for 40 minutes at 37°C and for 2 hours min at 25°C.
- Transformation: See transformation protocol.
USER enzyme:
USER™ (Uracil-Specific Excision Reagent) Enzyme generates a single nucleotide gap at the location of a uracil. USER Enzyme is a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII (New England biolabs® Inc.).
Protocol for Polymerase Chain Reaction Site-Directed Mutagenesis
We used PCR SDM to remove a Xbal restriction site from the luxCDABE cassette to make it compatible with the Biobrick system.
Primer design
Two complementary primers of 25-30 basepairs are designed.
The chosen sequence should be identical to the template strand, except for the single basepair which is to be mutated.
The melting temperature of the two primers are noted.
PCR reaction
Each sample reaction is prepared in a PCR-tube as indicated below:
- X µL of primer 1 for a total of 125 ng of DNA
- X µL of primer 2 for a total of 125 ng of DNA
- 1 µL of template dsDNA
- 10 µL of Phusion buffer (CONC???)
- 1 µL of dNTPs (CONC???)
- 0.5 µL of Phusion Hotstart enzyme (CONC???)
- X µL of ddH2O to a total volume of 50 microliters
Notes
- The template dsDNA should be complete plasmids obtained an purified from an overnight culture. The length of the plasmid should be known.
- A series of template dsDNA dilutions can be included in the range 5-50 ng of dsDNA per reaction. The remaining concentrations should be held constant.
- A control reaction containing ddH2O instead of template should be included.
| Temperature/°C |
Time/s |
Cycles |
| 98° |
30 |
12 |
| 98° |
10 |
12 |
| Average primer Tm +3 |
20 |
12 |
| 72° |
15 s/kb of plasmid length |
12 |
| 72° |
420 |
|
| 10° |
Remaining time |
|
Treatment towards transformation:
Dpn1 treatment:
- 0.5 µL of Dpn1 (20U/µL) is added directly to each PCR-tube, and the sample is gently mixed by pippeting up and down.
- Each sample is incubated at 37 °C for 60 minutes.
Transformation
See transformation protocol.
Optional:
Restriction site analysis.
Protocol for transformation in E. coli DH5α
The preparations for the transformation can preferably be done, while the USER cloning is incubating.
Materials
- LB plates (with antibiotics)
- E. coli DH5α competent cells
- Sterilized spatula
- USER reaction
Procedure:
- LB plates are taken out of the refrigerator and marked. Remember to use LB plates with the right antibiotics.
- 50 µL competent E. coli DH5α cells per USER reaction is taken from the -80C° freezer and place on ice. Additionally, 1,5 ml tubes are placed on ice.
- 5 µL USER reaction mix is added to the 50 µL competent E. coli DH5α cells. Mix well by pipetting.
- The cells are placed on ice for 30 min.
- The hot plate is set on 42 °C, and each transformation is heat shocked for 1 min. The cells are put directly on ice for 2 min afterwards.
- 200 µL recovery medium is added pr. cell tube.
- The cells are placed on hot plate on 300 rpm for 30 minutes.
- The hot plate is increased to 1000 rpm, and the cells are placed hereon for additional 1 hour.
Plating:
With antibiotic resistance gene.
- The transformed cells is transferred to an LB plate containing antibiotics and dispersed with the spatula.
- The transformed cells are incubated over night at 37°C.
- Next day; the plates are checked for visual colonies. These are cultivated.
Cultivation of tranformed cells:
Materials:
- LB Media
- Antibiotics (Matching the Resistance marker gene)
- Inoculation needle
Procedure:
- A couple of colonies are chosen from the LB-plate, and each colony is transferred to a small tube with 20 µL water.
- 5 µL of each colony is transferred to 5 mL LB + 5 µL antibiotics (in our case: chloramphenicol) with a pipet tip.
- Incubate over night at 37°C in the shaking incubator.
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