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
Luc14 KAH228 g034 1 0.302 0.151 2.003 301.829
Luc14 KAH228 g034 2 0.652 0.326 1.999 651.792
Luc14 KAH228 g034 3 0.449 0.223 2.009 448.873
Gal4 dox KAH228 g034 1 0.335 0.167 2 334.767
Gal4 dox KAH228 g034 2 0.333 0.166 2.006 333.159
Gal4 dox KAH228 g034 3 0.305 0.153 1.987 304.635



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
Gal4 puro g032 1 0.016 0.009 1.86 16.007 24.99 3.50 6.51
Gal4 puro g032 2 0.008 0.004 2.116 8.125 49.23
Gal4 puro g032 3 0.013 0.007 1.812 12.543 31.89 3.50 -0.39
Luc14 g044 1 0.015 0.008 1.763 14.617 27.37
Luc14 g044 2 0.009 0.006 1.552 9.059 44.15
Luc14 g044 3 0.056 0.03 1.864 56.272 7.11 1.50 6.39
Luc14 g046 1 0.11 0.058 1.877 109.619 3.65 1.50 9.85
Luc14 g046 2 0.094 0.05 1.877 93.996 4.26 1.50 9.24
Luc14 g046 3 0.102 0.055 1.843 101.658 3.93 1.50 9.57