Team:Copenhagen/Protocols

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

• 2,5 μl of each primer
• 4 μl template DNA
• 41 μl MasterMix

Colony PCR

Protocol for PCR reactions for individual genes

• 2.5 µl of each primer
• 0.5 µl template DNA
• 44.5 µl MasterMix

USER Cloning

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.

Protocol for Polymerase Chain Reaction Site-Directed Mutagenesis

• 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

• 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.

• 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.

Protocol for transformation in E. coli DH5α or E. Cloni

• LB plates (with antibiotics)
• E. coli DH5α (Bioline) or E. cloni-5α (Lucigen) competent cells
• Sterilized Gridalzky spatula
• USER reaction

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.

• 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.

• LB Media
• Antibiotics (Matching the Resistance marker gene)
• Inoculation needle

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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