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ASDF

Saturday 28^th September

Extraction of Genomic DNA, PCR Reaction and Restreak plates

Protocol: E. coli Colony PCR (pS006’, pS006*, pS013, pS004, pS004*, pS002*)

Extraction of Genomic DNA

1) Pick a single colony into 50 ul of H20.
Fresh colonies (grown that day) work best, but they can also come from 4 C.
Pipet 2ul onto plates to have C1-C8
Pipet 2 ul into 5ml Media with antibiotics to make liquid cultures

2) Boil for 5 minutes.
1.5 ul of this can be used directly for PCR.
Best if used directly, but can also be stored at 4 C for a few days.

PCR Reaction

Keep all the reagents at 4 °C while preparing the mixture.
Pre-heat the thermocycler to 95 °C and transfer your reaction directly from 4 °C.

Reagent	Volume	9x Volume
Forward Primer (10 uM)	0.5 µl	4.5 µl
Reverse Primer (10 uM)	0.5 µl	4.5 µl
Template DNA (from above)	1.5 µl	13.5 µl
Quick-Load® Taq 2X Master Mix	12.5 µl	112.5 µl9x Volume
Nuclease-free water	10 µl	90 µl
Total Volume	25 µl	125 µl

Thermocycler Protocol: Green Dream Taq
	Temp	Time
Start	95°C	30 sec	Melt
Cycle 1	95°C	15 sec	Melt	35 cycles
Cycle 2	46.8°C	30 sec	Anneal
Cycle 3	72°C	1 min per kb	Extend
Finish	72 °C	10 min	Extand
Store	10°C	Forever	Store

Restreak plates
Some of the plates were too full grown -> Rstreak for single colonies