Haynes Lab:Notebook/CRISPR Editing/2015/05/16

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05/16/2015

Cloning GFP behind Cas9
Will do ligation before and after purifying with Qiaquick PCR purification kit which will get rid of the 23 basepairs cut out of the backbone and the >10bps cut off the ends of the insert
before purification

50ng backbone (8486bp) = 50ng /(1000ng/50ul) = 2.5ul
10ng insert (824bp) = 10ng / (40ng/25ul) = 6.25ul

mixed backbone and insert, backbone only, brought both to 10ul with water
added 10ul 2x Roche ligation buffer
added 1ul Roche T4 ligase
mixed and spun
incubated at RT on benchtop for 15-20mins
moved 2ul of each reaction to a new tube, added 10ul water
water only control
added 50ul of my DH5αTs or 50ul of KAH's DH5αTs
incubate on ice for 30min
heat shock at 42°C for 35s
incubate on ice for 2min
add 750ul SOC to each tube
shake at 37°C for 1hour
plate all cells

Cell type plasmid 1 plasmid 2 rep 260 280 260/280 ng/µL ul for 400ng
Luc14 GFP g034 2 0.033 0.019 1.791 33.354 11.99256461
Luc14 GFP g034 3 0.027 0.014 1.953 26.814 14.91758037
Luc14 GFP KAH228 2 0.03 0.016 1.853 30.427 13.14621882
Luc14 GFP KAH228 3 0.033 0.016 2.096 32.56 12.28501229
Luc14 KAH228 g034 2 0.032 0.018 1.814 31.868 12.55177608
Luc14 KAH228 g034 3 0.032 0.017 1.869 31.64 12.64222503
Gal4-EED +dox GFP g034 2 0.033 0.017 1.916 33.466 11.95242933
Gal4-EED +dox GFP g034 3 0.027 0.014 1.953 26.521 15.08238754
Gal4-EED +dox GFP KAH228 2 0.03 0.017 1.75 30.466 13.12939014
Gal4-EED +dox GFP KAH228 3 0.023 0.012 1.964 22.794 17.54847767
Gal4-EED +dox KAH228 g034 2 0.025 0.013 1.935 24.575 16.27670397
Gal4-EED +dox KAH228 g034 3 0.029 0.015 1.925 29.369 13.61980319



cell type ug plasmid ul lipo rep 260 280 260/280 ng/µL ul for 400ng
Gal4-EED 0.5 3 1 0.034 0.018 1.91 33.545 11.92428082
Gal4-EED 0.5 3 2 0.031 0.017 1.886 31.165 12.83491096
Gal4-EED 1 3 1 0.03 0.015 1.939 29.994 13.33600053
Gal4-EED 1 3 2 0.031 0.018 1.721 30.674 13.04035991
Gal4-EED 0.5 5 1 0.028 0.015 1.943 28.44 14.06469761
Gal4-EED 0.5 5 2 0.027 0.014 1.921 27.34 14.63057791
Gal4-EED 1 5 1 0.03 0.016 1.884 30.303 13.2000132
Gal4-EED 1 5 2 0.029 0.015 1.938 28.813 13.88262243



' Cell type plasmid 1 plasmid 2 rep ng/µL ul for 400ng ul annealing buff ul water sur
1 Luc14 GFP g034 2 33.354 11.99 2.00 6.01 +
2 Luc14 GFP g034 3 26.814 14.92 2.00 3.08 +
3 Luc14 KAH228 g034 2 31.868 12.55 2.00 5.45 +
4 Luc14 KAH228 g034 3 31.64 12.64 2.00 5.36 +
5 Gal4-EED +dox GFP g034 2 33.466 11.95 2.00 6.05 +
6 Gal4-EED +dox GFP g034 3 26.521 15.08 2.00 2.92 +
7 Gal4-EED +dox KAH228 g034 2 24.575 16.28 2.00 1.72 +
8 Gal4-EED +dox KAH228 g034 3 29.369 13.62 2.00 4.38 +
9 Luc14 GFP KAH228 2 30.427 13.15 2.00 4.85 +
10 Luc14 KAH228 g034 2 31.868 12.55 2.00 5.45 -
11 Gal4-EED +dox KAH228 g034 2 24.575 16.28 2.00 1.72 -