Colony PCR for yeast 1-hybrid screen
1. Pipette 10ul 20mM NaOH into the appropriate number of wells in a 96-well
semi-skirted PCR plate for the number of colonies you wish to screen.
2. Pick a colony sized scoop of yeast from the patched plates using a pipette tip
and place tip in well containing NaOH. Leave tip in the well to track progress
(restreaked yeast can be up to 2 weeks old – if older replicate onto new
plates first to obtain fresh cells).
3. When all colonies have been picked gently shake the plate and then remove
all tips.
4. Seal plate with a heat seal.
5. Incubate at 99oC for 10 minutes in a PCR machine (plate can be kept for 2
weeks in the fridge).
6. Prepare the following PCR mix (volumes are for 100 reactions):
1.45ml
200ul
200ul
40ul
60ul
5ul
5ul
10ul
H2O
10x PCR buffer
Orange G dye
dNTPs (10mM)
MgCl2 (50mM)
SABR447 primer (100uM)
SABR448 primer (100uM)
Taq polymerase
7. Add 19ul PCR mix to each well in a fresh semi-skirted PCR plate.
8. Add 1.2ul yeast colony mix to each well.
9. Run the following PCR reaction:
94oC
94oC
55oC
72oC
72oC
4oC
2 minutes
30 seconds
30 seconds
3 minutes
7 minutes
10 minutes
40 cycles
10. Run 3ul of each reaction of pre-cast agarose gels (0.75XTAE)
11. If the majority of reactions have worked then clean up the remainder of each
reaction using a 96-well PCR cleanup plate.