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

From OpenWetWare
Jump to navigationJump to search
Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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


Ran 6 ul on gel.

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

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)

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


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


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/CRISPR_Editing/2014/09/15&oldid=820522"