Add one ml overnight culture to a cryotube from step 1
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)
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Preparation of Cam stock solution
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Recipe on M9 minimal media
Make M9 salts
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
Measure ~700ml of distilled H2O (sterile)
Add 200ml of M9 salts
Add 2ml of 1M MgSO4 (sterile)
Add 20 ml of 20% glucose (or other carbon source)
Add 100ul of 1M CaCl2 (sterile)
Adjust to 1000ml with distilled H2O
Restriction analysis
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
Divide the mastermix into tubes and add DNA to a final concentration of 10 ng/μl
Incubate the reaction mix at the restriction enzymes optimal temperature for 30-60 minutes
Add loading buffer to samples and run on 1% agarose gel with ethidium bromide 0.2 μg/ml