Haynes Lab:Notebook/CRISPR Editing/2015/11/08

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11/08/2015

Nested PCR of g032 Luc14 samples (last run had a band in the negative ctrl lane)
Ran on gel. light band in neg ctrl lane. Purified anyway.

Cell type gRNA rep 260 280 260/280 ng/µL ul for 400ng ul ann buff ul water
Luc14g03210.0770.0421.84977.1745.181.508.32
Luc14g03220.0830.0441.89883.2644.801.508.70
Luc14g03230.0940.0521.80693.8264.261.509.24



Cut 100ng of each HDR sample with EcoRI.
Controls

Donor PCR products
Unedited Luc14 gDNA PCR w P197/198
No enzyme ctrl for each

Total of 9 with enzyme and 9 without

Cell type gRNA donor rep ng/µL ul for 100ng water
Luc14g031g031 EcoRI135.132.851.15
Luc14g031g031 EcoRI265.8721.522.48
Luc14g031g031 EcoRI358.1611.722.28
N/AN/Ag031 EcoRIN/A63.71.572.43
Luc14g034g034 EcoRI136.1152.771.23
Luc14g034g034 EcoRI228.5073.510.49
Luc14g034g034 EcoRI337.642.661.34
N/AN/Ag034 EcoRIN/A1320.763.24
Luc14N/AN/AN/A1210.833.17

With enzyme MM

' 1 rxn x10
FD buffer110
EcoRI0.55
Water4.545
Total6

Without enzyme MM

' 1 rxn x10
FD buffer110
EcoRI00
Water550
Total6

Incubate at 37°C for 15 minutes, heat inactivate at 80°C for 5 minutes.
Dilute 1:10, run on BA.


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