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
1 Luc14 g023 1 P173
2 Luc14 g023 2 P173
3 Luc14 g023 3 P173
4 Gal4 g023 1 P173
5 Gal4 g023 2 P173
6 Gal4 g023 3 P173
7 Luc14 untreated P173
8 Luc14 untreated P173
Sample name template Primer
9 Luc14 g034 1 P176
10 Luc14 g034 2 P176
11 Luc14 g034 3 P176
12 Gal4 g034 1 P176
13 Gal4 g034 2 P176
14 Gal4 g034 3 P176
15 Luc14 untreated P176
16 Luc14 untreated P176



PCR of gDNA samples from gRNA treated samples:

Cell type treatment rep ng/µL ul for 200ng 2.1rxns
Luc14 g023 1 82.358 2.43 5.10
Luc14 g023 2 76.437 2.62 5.50
Luc14 g023 3 119.281 1.68 3.53
GAL4-EED +dox g023 1 122.844 1.63 3.42
GAL4-EED +dox g023 2 102.434 1.95 4.10
GAL4-EED +dox g023 3 101.862 1.96 4.12
Luc14 g045 1 181.634 1.10 2.31
Luc14 g045 2 246.109 0.81 1.71
Luc14 g045 3 174.603 1.15 2.41
GAL4-EED +dox g045 1 74.461 2.69 5.64
GAL4-EED +dox g045 2 82.614 2.42 5.08
GAL4-EED +dox g045 3 58.964 3.39 7.12
Luc14 g046 1 255.228 0.78 1.65
Luc14 g046 2 170.592 1.17 2.46
Luc14 g046 3 144.251 1.39 2.91
GAL4-EED +dox g046 1 72.999 2.74 5.75
GAL4-EED +dox g046 2 78.598 2.54 5.34
GAL4-EED +dox g046 3 77.323 2.59 5.43
Luc14 g048 1 172.85 1.16 2.43
Luc14 g048 2 217.50 0.92 1.93
Luc14 g048 3 190.13 1.05 2.21
GAL4-EED +dox g048 1 183.71 1.09 2.29
GAL4-EED +dox g048 2 189.83 1.05 2.21
GAL4-EED +dox g048 3 209.10 0.96 2.01
Luc14 Untreated 1 203.77 0.98 2.06
Luc14 Untreated 2 201.30 0.99 2.09
Luc14 Untreated 3 221.91 0.90 1.89
GAL4-EED +dox Untreated 1 205.61 0.97 2.04
GAL4-EED +dox Untreated 2 219.95 0.91 1.91
GAL4-EED +dox Untreated 3 205.91 0.97 2.04


Mastermix 1

' 1rxn 50.6
H20 30.8 1558.48
HF 5X MasterMix 10 506
dNTPs 1 50.6
FP P197 2.5 126.5
RP P198 2.5 126.5
DMSO 1.5 75.9
Total 48.3 2443.98

Mastermix 2

' 1rxn 2.1 rxns
MM1 47.3 101.43
Template 2 4.2
Phusion 0.5 1.05
Total 50

Sending out for sequencing

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