Haynes Lab:Notebook/CRISPR Editing/2015/02/06

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02/06/2015

Trying Surveyor on g023 with a shorter transcript. Still probably too long but I know these primers work and it's still like half of the transcript I was using before. First, PCR with phusion:

' 1rxn 6.2
H20 30.8 190.96
HF 5X MasterMix 10 62
dNTPs 0.2 1.24
FP P159 2.5 15.5
RP P170 2.5 15.5
Total 46 285.2
Cell type treatment rep ng/µL ul for 200ng
Luc14 g023 1 82.358 2.43
Luc14 g023 2 76.437 2.62
Luc14 g023 3 119.281 1.68
GAL4-EED +dox g023 1 122.844 1.63
GAL4-EED +dox g023 2 102.434 1.95
GAL4-EED +dox g023 3 101.862 1.96
' 1rxn
MM1 46
Template 2
DMSO 1.5
Phusion 0.5
Total 50

PCR

98°C for 3 min
30 cyles
98°C for 10s
69°C for 30s
72°C for 30s
72°C for 10 mins
4°C