Team:DTU-Denmark/Methods/Protocols

1. Add 1 ml sterile 50% glycerol to a cryotube.
2. Add 1 ml overnight culture to a cryotube from step 1
3. Store at -80 °C

• Make a mastermix of the reagents that are common for all reactions. Make enough for 0,5 extra reaction. Add reagents in the order they are listed above.
• The polymerase stock must only be taken out of the freezer when needed and must be put back immediately after use.
• Remember to mix thoroughly before aliquotting to separate PCR tubes, and try not to make any bubbles. Reverse pipetting works well for aliquoting.
• Mark the PCR tubes on lid and side and place in thermocycler.
• The annealing temperature should match the primer melting temperatures minus 5 degrees. This can be hard since Tm predictors tend to disagree.
• Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).

1. Make M9 salts
2. To make M9 Salts aliquot 800ml H2O and add
• 64g Na2HPO4-7H2O
• 15g KH2PO4
• 2.5g NaCl
• 5.0g NH4Cl
• Stir until dissolved
• Adjust to 1000ml with distilled H2O
• Sterilize by autoclaving
3. Measure ~700ml of distilled H2O (sterile)
4. Add 200ml of M9 salts
5. Add 2ml of 1M MgSO4 (sterile)
6. Add 20 ml of 20% glucose (or other carbon source)
7. Add 100ul of 1M CaCl2 (sterile)
8. Adjust to 1000ml with distilled H2O

1. Make a mastermix containing.
• 1X NEBuffer (according to the desired restriction enzyme)
• 1X BSA (note if NEBuffer is called X.1 BSA is already added in the buffer)
• 1 unit/μl restriction enzyme
2. Divide the mastermix into tubes and add DNA to a final concentration of 10 ng/μl
3. Incubate the reaction mix at the restriction enzymes optimal temperature for 30-60 minutes
4. Add loading buffer to samples and run on 1% agarose gel with ethidium bromide 0.2 μg/ml
5. Analyze your gel

Transformation with Chemically competent E. coli

IMPORTANT.! Keep everything on ice unless otherwise stated in the protocol.

1. Competent cells are defrosted in ice bath.
2. 40 μl cells is transferred to a cooled eppendorf tube.
3. 2 μl ice cold DNA is transferred to the competent cells, DNA and cells is mixed by inverting the tube 2-3 times.
4. DNA and cell mix is kept on ice for 10 minutes.
5. Cells are heat shocked at 42 °C for 30 seconds in water bath for optimal heat transfer.
6. Cells are kept on ice for 3 minutes.
7. Add 500 μl LB with no antibiotics and incubate cells at 37 °C for 30-60 minutes depending on antibiotic marker
8. Plate cells on LB plate with required antibiotic and incubate overnight at 37 °C.

Transformation with electro-competent E. coli

IMPORTANT.! Keep everything on ice unless otherwise stated in protocol.

Preparation: Put electro cuvettes, LB media with no antibiotics and DNA on ice in order to cool it down.

1. Defrost electro-competent cells on ice
2. Transfer 1 μl DNA to the competent cells
3. Transfer DNA and cell mix to electroporation cuvette
4. When ready the cuvette is quickly dried with paper towel and inserted to the electroporater and a shock is given. Immediately after the shock is given transfer 1 ml of ice cold LB to the cuvette and transfer the cells and media from the cuvette to an eppendorf tube and put the tube on ice.
5. Leave the cells on ice for 10 minutes.
6. Incubate the cells at 37 °C for 30-60 minutes depending on antibiotic marker
7. Plate 100 μl on a half plate of LB with required antibiotic
8. Spin the cells briefly at maximum RPM and decant the supernatant gently, approximately 100 μl will remain in the tube, resuspend the pellet in the rest of the supernatant and plate it on the other half of the plate used in step 7.
9. Incubate plates at 37 °C overnight