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.