Haynes Lab:Notebook/CRISPR Editing/2015/02/13

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02/13/2015

Want to sequence to determine editing efficiencies for all samples. Will prep the ones I have PCRd already, g023 and g034:

g023 and P173, distance 150
g034 and P176, distance 291
Sample name template Primer
1Luc14 g023 1P173
2Luc14 g023 2P173
3Luc14 g023 3P173
4Gal4 g023 1P173
5Gal4 g023 2P173
6Gal4 g023 3P173
7Luc14 untreatedP173
8Luc14 untreatedP173
Sample name template Primer
9Luc14 g034 1P176
10Luc14 g034 2P176
11Luc14 g034 3P176
12Gal4 g034 1P176
13Gal4 g034 2P176
14Gal4 g034 3P176
15Luc14 untreatedP176
16Luc14 untreatedP176



PCR of gDNA samples from gRNA treated samples:

Cell type treatment rep ng/┬ÁL ul for 200ng 2.1rxns
Luc14g023182.3582.435.10
Luc14g023276.4372.625.50
Luc14g0233119.2811.683.53
GAL4-EED +doxg0231122.8441.633.42
GAL4-EED +doxg0232102.4341.954.10
GAL4-EED +doxg0233101.8621.964.12
Luc14g0451181.6341.102.31
Luc14g0452246.1090.811.71
Luc14g0453174.6031.152.41
GAL4-EED +doxg045174.4612.695.64
GAL4-EED +doxg045282.6142.425.08
GAL4-EED +doxg045358.9643.397.12
Luc14g0461255.2280.781.65
Luc14g0462170.5921.172.46
Luc14g0463144.2511.392.91
GAL4-EED +doxg046172.9992.745.75
GAL4-EED +doxg046278.5982.545.34
GAL4-EED +doxg046377.3232.595.43
Luc14g0481172.851.162.43
Luc14g0482217.500.921.93
Luc14g0483190.131.052.21
GAL4-EED +doxg0481183.711.092.29
GAL4-EED +doxg0482189.831.052.21
GAL4-EED +doxg0483209.100.962.01
Luc14Untreated1203.770.982.06
Luc14Untreated2201.300.992.09
Luc14Untreated3221.910.901.89
GAL4-EED +doxUntreated1205.610.972.04
GAL4-EED +doxUntreated2219.950.911.91
GAL4-EED +doxUntreated3205.910.972.04


Mastermix 1

' 1rxn 50.6
H2030.81558.48
HF 5X MasterMix10506
dNTPs150.6
FP P1972.5126.5
RP P1982.5126.5
DMSO1.575.9
Total48.32443.98

Mastermix 2

' 1rxn 2.1 rxns
MM147.3101.43
Template24.2
Phusion0.51.05
Total50

Sending out for sequencing

Sample name template Primer
17Luc14 g023 1P149
18Luc14 g023 2P149
19Luc14 g023 3P149
20Gal4 g023 1P149
21Gal4 g023 2P149
22Gal4 g023 3P149
23Luc14 g045 1P176
24Luc14 g045 2P176
25Luc14 g045 3P176
26Gal4 g045 1P176
27Gal4 g045 2P176
28Gal4 g045 3P176
29Luc14 g046 1P165
30Luc14 g046 2P165
31Luc14 g046 3P165
32Gal4 g046 1P165
33Gal4 g046 2P165
34Gal4 g046 3P165
35Luc14 g048 1P170
36Luc14 g048 2P170
37Luc14 g048 3P170
38Gal4 g048 1P170
39Gal4 g048 2P170
40Gal4 g048 3P170
41Luc14 Unt 1P149
42Luc14 Unt 2P149
43Luc14 Unt 3P149
44Gal4 Unt 1P149
45Gal4 Unt 2P149
46Gal4 Unt 3P149
47Luc14 Unt 1P176
48Luc14 Unt 2P176
49Luc14 Unt 3P176
50Gal4 Unt 1P176
51Gal4 Unt 2P176
52Gal4 Unt 3P176
53Luc14 Unt 1P165
54Luc14 Unt 2P165
55Luc14 Unt 3P165
56Gal4 Unt 1P165
57Gal4 Unt 2P165
58Gal4 Unt 3P165
59Luc14 Unt 1P170
60Luc14 Unt 2P170
61Luc14 Unt 3P170
62Gal4 Unt 1P170
63Gal4 Unt 2P170
64Gal4 Unt 3P170



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