Haynes Lab:Notebook/CRISPR Editing/2015/05/22

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05/22/2015

SYBR qPCR to determine plasmid counts for dilution experiment

pX330 primer P209/210 (G)
internal primer 18S (C)
Dilution 2ng/rxn

Step 1: make two PCR tubes for each sample with the following volumes of water and gDNA for 2ng of gDNA per well

' 1rxn x3.5 = 7ng
gDNA x
water to 4.5
Total 4.5 15.75
Tube label Cell type gRNA ug DNA ul lipo rep ng/µL dilute 1:20 ul dilution ul water
1 Gal4-EED g034 0.5 3 1 81.058 4.0529 1.73 14.02
2 Gal4-EED g034 0.5 3 2 86.697 4.33485 1.61 14.14
3 Gal4-EED g034 0.5 5 1 23.89 1.1945 5.86 9.89
4 Gal4-EED g034 0.5 5 2 24.767 1.23835 5.65 10.10
5 Gal4-EED g034 1 3 1 85.32 4.266 1.64 14.11
6 Gal4-EED g034 1 3 2 70.244 3.5122 1.99 13.76
7 Gal4-EED g034 1 5 1 13.387 0.66935 10.46 5.29
8 Gal4-EED g034 1 5 2 23.213 1.16065 6.03 9.72
9 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

8 samples, 2 tubes/sample, 1 tube for no template control, total of 17 tubes
' 1rxn 3.5/triplicate tube 17.7
primer 3 10.5 185.85
SYBR 7.5 26.25 464.625
Total 10.5 36.75 650.475

Step 3: mix gDNA and primer mixes

Add 36.75ul primer mix to each sample tube

Step 4: pipette plate

mix tubes immediately before pipetting, add 15 ul from each tube into 3 wells
' 1-3 4-6 7-9 10-12
A __1 G__ __1 C__ blank-G ______
B __2 G__ __2 C__ blank-C ______
C __3 G__ __3 C__ ______ ______
D __4 G__ __4 C__ ______ ______
E __5 G__ __5 C__ ______ ______
F __6 G__ __6 C__ ______ ______
G __7 G__ __7 C__ ______ ______
H __8 G__ __8 C__ ______ ______




Cell type Plasmid 1 Plasmid 2 Rep 260 280 260/280 ng/µL 1:20 dil ul dil for 7ng ul water
Luc14 KAH226 g034 1 0.081 0.04 2.021 80.531 4.02655 1.738460965 14.01153903
Luc14 KAH226 g034 2 0.126 0.061 2.06 126.451 6.32255 1.107148223 14.64285178
Luc14 KAH226 g034 3 0.091 0.044 2.045 90.862 4.5431 1.540798133 14.20920187
Luc14 GFP g034 1 0.104 0.051 2.052 104.239 5.21195 1.343067374 14.40693263
Luc14 GFP g034 2 0.065 0.031 2.094 64.965 3.24825 2.155006542 13.59499346
Luc14 GFP g034 3 0.039 0.02 1.99 39.096 1.9548 3.580928995 12.169071
15.75