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.
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'
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
|
|