Haynes Lab:Notebook/CRISPR Editing/2014/09/15

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09/15/2014

Purified 'gRNA 20 +dox rep 1' PCR reaction with P197/P198, SURVEYOR Ctrl C PCR reaction, and SURVEYOR Ctrl G PCR reaction. Ran binding through twice and water elution through twice. Eluted in 30ul.

Sample Read# 260 280 260/280 ng/µL
gRNA 20 +dox rep 1' PCR reaction with P197/P198 0.003 0.001 3.444 3.212
SURVEYOR Ctrl C PCR reaction 0.066 0.035 1.9 65.558
SURVEYOR Ctrl G PCR reaction 0.053 0.028 1.877 53.041


After looking at the concentration of the sample DNA (3.212 with 260/280 of 3.4), I'd expect to not see anything on the gel but I see a decent band (lane 1)
Measured the concentration again, not sure what to make of this

Sample Read# 260 280 260/280 ng/µL
Nothing 0.005 0.003 1.643 4.841
gRNA 20 PCR 0.008 0.004 2.222 8.29
gRNA 20 PCR 0.014 0.007 1.928 13.619


Ran 6 ul on gel.
14.09.15 SRVYR control gel.png

Lane Sample Band size
1 Purified 'gRNA 20 +dox rep 1' PCR reaction with P197/P198 1660
2 SURVEYOR Ctrl C PCR reaction
3 SURVEYOR Ctrl G PCR reaction



'Ran gel of all the Luc->AmCyan donor PCR reactions'

14.09.15 PCR of Luc to AmCyan gel.png


Lane Sample Band size
1 pure 1 729
2* 2a 764
3 2b 789
4 pure 3a 834
5 pure 3b 877
6 4a 962
7 4b 962
8 pure 4c 918
9 4c3 918
  • in oww notebook, I wrote that I used P185, which shouldn't amplify this because it should amplify mCh. This length is assuming I used P186


Looking back at the PCR I ran for the fourth round, the primers don't overlap with the templates so it makes sense that I didn't get the amplicons I was expecting. I am re-running round four with primers that actually overlap.

Sample Template F primer R primer At length
4e 3a (pure) P178 P181 65 877