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Saturday 28th 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