Team:DTU-Denmarkgogo

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Protocols

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

PCR
  • 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. This can be hard as Tm predictors tend to disagree.
  • Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).

Casting of gels and gel electrophoresis (remember to include EtBr)
  1. A
  2. A
  3. S

Preparation of Cam stock solution
  1. h
  2. u
  3. i

Recipe on M9 minimal media
  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

Restriction analysis
  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

Rasmus Bech

Thor Bech Johannesen

Overview

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