Haynes Lab:Notebook/CRISPR Editing/2016/05/27

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05/27/2016

Ran g032, g044, g046 P197/198 PCR from yesterday on gel. G032 looked great, g044 and 46 were very faint. Ran nested PCR of all of them. G032 diluted 1:1000, dox with my phusion, puro with braflab phusion, 44 and 46 diluted 3:1000 with braflab phusion.

Extracted gDNA from KAH228/g034 cells:

Cell type drug re-opener gRNA rep 260 280 260/280 ng/µL
Luc14KAH228g03410.3020.1512.003301.829
Luc14KAH228g03420.6520.3261.999651.792
Luc14KAH228g03430.4490.2232.009448.873
Gal4doxKAH228g03410.3350.1672334.767
Gal4doxKAH228g03420.3330.1662.006333.159
Gal4doxKAH228g03430.3050.1531.987304.635



Nested PCR of g032, g044, g046

Gal4 +puro g032 had faint bands, no band in neg ctrl, decided to purify
Gal4 +dox g034 had no bands, my phusion is bad
Luc14 g044 faint bands, no band in neg ctrl, purified
Luc14 g046 super bright bands, no band in neg ctrl


Cell type drug gRNA rep 260 280 260/280 ng/µL ul for 400ng ul ann buff ul H2O
Gal4purog03210.0160.0091.8616.00724.993.506.51
Gal4purog03220.0080.0042.1168.12549.23
Gal4purog03230.0130.0071.81212.54331.893.50-0.39
Luc14g04410.0150.0081.76314.61727.37
Luc14g04420.0090.0061.5529.05944.15
Luc14g04430.0560.031.86456.2727.111.506.39
Luc14g04610.110.0581.877109.6193.651.509.85
Luc14g04620.0940.051.87793.9964.261.509.24
Luc14g04630.1020.0551.843101.6583.931.509.57


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