Haynes Lab:Notebook/CRISPR Editing/2015/05/27

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05/27/2015

Redo qPCR with the right control primers
Dilutions for 2ng gDNA per well:

Tube label Cell type gRNA plasmid 2 ug DNA ul lipo rep ng/µL dilute 1:20 ul dilution ul water
1 Gal4-EED g034 - 0.5 3 1 81.058 4.05 1.73 14.02
2 Gal4-EED g034 - 0.5 3 2 86.697 4.33 1.61 14.14
3 Gal4-EED g034 - 0.5 5 1 23.89 1.19 5.86 9.89
4 Gal4-EED g034 - 0.5 5 2 24.767 1.24 5.65 10.10
5 Gal4-EED g034 - 1 3 1 85.32 4.27 1.64 14.11
6 Gal4-EED g034 - 1 3 2 70.244 3.51 1.99 13.76
7 Gal4-EED g034 - 1 5 1 13.387 0.67 10.46 5.29
8 Gal4-EED g034 - 1 5 2 23.213 1.16 6.03 9.72
9 Luc14 g034 GFP - - 1 104.239 5.21 1.34 14.41
10 Luc14 g034 GFP - - 2 64.965 3.25 2.16 13.59
11 Luc14 g034 GFP - - 3 39.096 1.95 3.58 12.17
12 Gal4-EED+dox g034 GFP - - 1 160 8 0.88 14.88
13 Gal4-EED+dox g034 GFP - - 2 129 6.45 1.09 14.66
14 Gal4-EED+dox g034 GFP - - 3 132 6.6 1.06 14.69
15 water - - - - - - - - 15.75



Step 2: make primer master mixes.
Will have to add 3.5x to each triplicate gDNA tube so can calculate mastermixes from that
14 samples, 1 tube for no template control, total of 15 tubes per primer.

' 1rxn 3.5/triplicate tube 15.7
primer 3 10.5 164.85
SYBR 7.5 26.25 412.125
Total 10.5 36.75 576.975

Make these mixes for each primer pair, add 36.75 to each triplicate tube, then pipette 15ul into 3 well of the plate.


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