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
Luc14 g032 1 0.077 0.042 1.849 77.174 5.18 1.50 8.32
Luc14 g032 2 0.083 0.044 1.898 83.264 4.80 1.50 8.70
Luc14 g032 3 0.094 0.052 1.806 93.826 4.26 1.50 9.24



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
Luc14 g031 g031 EcoRI 1 35.13 2.85 1.15
Luc14 g031 g031 EcoRI 2 65.872 1.52 2.48
Luc14 g031 g031 EcoRI 3 58.161 1.72 2.28
N/A N/A g031 EcoRI N/A 63.7 1.57 2.43
Luc14 g034 g034 EcoRI 1 36.115 2.77 1.23
Luc14 g034 g034 EcoRI 2 28.507 3.51 0.49
Luc14 g034 g034 EcoRI 3 37.64 2.66 1.34
N/A N/A g034 EcoRI N/A 132 0.76 3.24
Luc14 N/A N/A N/A 121 0.83 3.17

With enzyme MM

' 1 rxn x10
FD buffer 1 10
EcoRI 0.5 5
Water 4.5 45
Total 6

Without enzyme MM

' 1 rxn x10
FD buffer 1 10
EcoRI 0 0
Water 5 50
Total 6

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