User:Cassandra M Barrett/Notebook/Haynes Lab/2015/08/31

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MV11+T2A-EGFP colony PCR

Purpose: 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:

15.09.01colony PCR .jpg

The absolutely ginormous gel image shows both weak and strong positives for all colonies screened. All bands are around the desired 849bp as indicated by the positive control. There is some very slight banding on the negative control indicating possible contamination.

Strong positives 4,9, and 13 were chosen to be miniprepped. After growth on spot plate, each of these colonies was used to inoculate separate tubes of 3mL LB+Amp, which were grown shaking overnight at 37C. Cultures were miniprepped the following day.Concentrations are as follows:

Colony 4: 83 ng/uL Colony 9: 51 ng/uL Colony 13: 45 ng/uL

Next Step: Sequence verify construct