User:Cassandra M Barrett/Notebook/Haynes Lab/2015/08/31: Difference between revisions
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Methods: | Methods: | ||
14 colonies picked, | 14 colonies picked for PCR, - control (water only), + control (pX330g) | ||
Primers: P231/232 | |||
Create PCR mastermix: | |||
1 reaction: | |||
2XGoTaq Mix 5uL | |||
FP 0.5uL | |||
RP 0.5uL | |||
Water 4uL | |||
18X MM made for 16 reactions | |||
10uL aliquoted per tube | |||
Colonies picked with pipette tip and incubated in mix for 10min at RT, 2uL of each control substance added to appropriate tubes | |||
Tips removed and spotted onto LB+Amp, incubated overnight at 37C | |||
PCR run using GoTaq protocol (60C, 30sec) | |||
Results: | |||
Revision as of 11:50, 4 September 2015
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MV11+T2A-EGFP colony PCRPurpose: to confirm success of transformation with ligated MV11+T2A-EGFP construct Use same primers as used for amplification to verify proper insertion of fragment into vector, vector already verified by ability to grow on Amp Methods: 14 colonies picked for PCR, - control (water only), + control (pX330g) Primers: P231/232 Create PCR mastermix: 1 reaction: 2XGoTaq Mix 5uL FP 0.5uL RP 0.5uL Water 4uL 18X MM made for 16 reactions 10uL aliquoted per tube Colonies picked with pipette tip and incubated in mix for 10min at RT, 2uL of each control substance added to appropriate tubes Tips removed and spotted onto LB+Amp, incubated overnight at 37C PCR run using GoTaq protocol (60C, 30sec) Results:
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