Colony PCR

Colony PCR

Colony PCR

1. Master mix (for 96 well plate):

H2O 7.6 ml

Tag 200 ul

PCR buffer(10X) 1 ml

dNTPs (10X) 1 ml

Primers 5' (100uM) 100ul

3' (100uM) 100ul

aliquot into 96-well plate at 50 ul/tube

2. Fresh grown colonies (or 5ul our of 2ml of fresh grown bacterial culture) into 100 ul H2O, Heat at 100 C, 5’ (or boil 5’)

3. 5 ul of heated colony suspension into PCR tube containing 25 ul rxn mix.

4. PCR

95 C 5 min

94 C 30``

55 C 30``

72 C 1` (for products between 200-1kb)

30 X

5. Loading 1% metaphor agrose gel with DNA ladders.