Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/01

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Overview

QuikChange transformation results:

  • Negative control (QuikChange on pWhitescript, no DpnI treatment): about 350 colonies, mostly white, maybe 25 blue
  • positive control (QuikChange on pWhitescript, DpnI treated): 34 colonies, 23 blue
  • EM7:ZeoR_v0120: about 2 dozen colonies
  • MV8: about 1 dozen colonies

QuikChange efficiency is about 68%. Picked 8 colonies from each sample plate and set up liquid cultures and streak plates. Today I'm performing 'colony' PCR on the streak plates and then running them on a gel. For the samples that look good (have product in the right size range), I'll do PCR cleanup and submit for sequencing, as well as perform plasmid preps on the pellets of the liquid cultures.

Gel Verification

File:2016-02-01 QuikChange colony PCR.tif

For EM7:ZeoR_v0120, colonies 1, 2, 3, 4, 5, and 7 have the correct size insert (~700 bp). For MV8, colonies 2, 3, 5, 6, 7, and 8 have the correct size insert (~440 bp). Will perform PCR clean-up on all successful PCR products and send 3 from each type of plasmid out for sequencing by the core lab.

DNA Quantification

Sample ID 260/280 ng/µL
EM7:ZeoR post PCR 1 2 23.764
EM7:ZeoR post PCR 2 1.968 32.639
EM7:ZeoR post PCR 3 2.092 24.056
EM7:ZeoR post PCR 4 1.864 24.298
EM7:ZeoR post PCR 5 1.991 23.82
EM7:ZeoR post PCR 7 1.877 22.107
MV8 post PCR 2 1.88 45.936
MV8 post PCR 3 1.87 51.642
MV8 post PCR 5 1.872 54.518
MV8 post PCR 6 1.907 48.089
MV8 post PCR 7 1.912 47.318
MV8 post PCR 8 1.939 44.961

Samples for Sequencing

Sample ID Primer
EM7:ZeoR post PCR 1 P0001
EM7:ZeoR post PCR 2 P0001
EM7:ZeoR post PCR 3 P0001
MV8 post PCR 2 EM7_BB_fwd
MV8 post PCR 3 EM7_BB_fwd
MV8 post PCR 5 EM7_BB_fwd

Sequencing Results (2016-02-03)

Got the sequences back for the MV8 samples, they all look terrible. Going to try miniprepping and submitting plasmids instead.