Team:Copenhagen/Protocols

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Primer design for point mutation in Lux Casette

  • The Lux cassette of 5798 bp had a Xbal restriction site that was removed by designing to primers of 25-30 base pairs with overlapping sequences. This sequence was designed as a USER site that covered the Xbal restriction site. The chosen USER site sequence should be identical to the template strand, except for the single base pair, which is to be mutated.
  • Two primers to each end of the Lux cassette were also designed.
  • Two PCR reactions were carried out with two sets of primers. One set each containing a primer annealing to the 3’ and a primer containing the new USER site sequence. And an other set containing a primer annealing the 5’ end respectively together with the complementary primer encoding the new USER site sequence.

PCR reaction for amplification of backbone pSB1C3

The following protocol is used to amplify the pSB1C3 backbone vector.
To each PCR tube the following is added:
  • 2.5 µl of each primer
  • 4 µl template DNA
  • 41 µl MasterMix


Master Mix
10 mM X7 Buffer
10 mM dNTP's
2 mM X7 DNA polymerase (Produced in our lab)

The PCR programme used:
StepTemperature/°CTimeCycles
Int. Denaturation 95° 120 sec
Denaturation 95° 30 sec 32
Tm 55° 12 sec 32
Elongation 68° 1.25 min 32
Final extension 68° 10 min
Hold 10°

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 MangoMix™ (5 mM) (Bioline)

MangoMix™
MangoTaq DNA polymerase
Orange reference dye
MgCl2
dNTP

The PCR programme used:
StepTemperature/°CTimeCycles
Int. Denaturation 96° 3 min
Denaturation 96° 20 sec 25
Tm 60° 20 sec 25
Elongation 72° * sec 25
Final extension 72° 7 min
Hold 10°
*= elongation time for MangoTaq DNA polymerase should be calculated so it is compatible with the size of the template. Taq writes 1kb/60sec.

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


Master Mix
10 mM X7 Buffer
10 mM dNTP's
2 mM X7 DNA polymerase (Produced in our lab)

The PCR programme used:
StepTemperature/°CTime/SecCycles
Int. Denaturation 98° 30 sec
Denaturation 98° 10 sec 32
Tm 60° 20 sec 32
Elongation 72° * sec 32
Final extension 72° 7 min
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.


USER Cloning


Procedure:
  1. The PCR product must be purified from a gel before (GenEluteTM HP plasmid MiniPrep kit (Sigma-Aldrich)) used in USER cloning.
  2. 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.)
  3. 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
  4. The USER mix is transferred to each Eppendorf tube and incubated for 2 hours at 37°C.
  5. 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.
  6. 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 transformation in E. coli DH5α or E. Cloni

The preparations for the transformation can preferably be done, while the USER cloning is incubating.
Materials
  • E. coli DH5α (Bioline) or E. cloni-5α (Lucigen) competent cells
  • USER reaction

Procedure:
  1. LB plates are taken out of the refrigerator and marked. Remember to use LB plates with the right antibiotics.
  2. 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.
  3. 5 µL USER reaction mix is added to the 50 µL competent E. coli DH5α cells. Mix well by pipetting.
  4. The cells are placed on ice for 30 min.
  5. 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.
  6. 200 µL recovery medium is added pr. cell tube.
  7. The cells are placed on hot plate on 300 rpm for 30 minutes.
  8. The hot plate is increased to 1000 rpm, and the cells are placed hereon for additional 1 hour.

Plating on LB plates: Materials
  • LB plates (with antibiotics)
  • Transformed E. coli cells
  • Sterilized Gridalzky spatula
Procedure
  • The transformed cells is transferred to an LB plate containing antibiotics and dispersed with the Drigalski 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:
  1. A couple of colonies are chosen from the LB-plate, and each colony is transferred to a small tube with 20 µL water.
  2. 5 µL of each colony is transferred to 5 mL LB + 5 µL antibiotics (in our case: chloramphenicol) with a pipet tip.
  3. Incubate over night at 37°C in the shaking incubator.

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