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/P1980.0030.0013.4443.212
SURVEYOR Ctrl C PCR reaction0.0660.0351.965.558
SURVEYOR Ctrl G PCR reaction0.0530.0281.87753.041


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
Nothing0.0050.0031.6434.841
gRNA 20 PCR0.0080.0042.2228.29
gRNA 20 PCR0.0140.0071.92813.619


Ran 6 ul on gel.

Lane Sample Band size
1Purified 'gRNA 20 +dox rep 1' PCR reaction with P197/P1981660
2SURVEYOR Ctrl C PCR reaction
3SURVEYOR Ctrl G PCR reaction



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


Lane Sample Band size
1pure 1729
2*2a764
32b789
4pure 3a834
5pure 3b877
64a962
74b962
8pure 4c918
94c3918
  • 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
4e3a (pure)P178P18165877



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