07/20/2015
Miniprep and sequence new gRNA plasmids
Sent out for sequencing with P139. Made a streak plate and seeded more 6mL cultures for each
| gRNA
|
260
|
280
|
260/280
|
ng/µL
|
| gn001 T |
0.258 |
0.133 |
1.945 |
258.246
|
| gn001 B |
0.3 |
0.156 |
1.919 |
299.748
|
| gn003 T |
0.295 |
0.153 |
1.93 |
294.921
|
| gn003 B |
0.256 |
0.134 |
1.918 |
256.118
|
| g005 |
0.269 |
0.14 |
1.917 |
269.101
|
| g0013 |
0.209 |
0.11 |
1.891 |
208.912
|
| g057 |
0.226 |
0.118 |
1.916 |
225.742
|
| g058 |
0.242 |
0.126 |
1.913 |
241.86
|
| g059 |
0.205 |
0.107 |
1.916 |
205.376
|
| g060 |
0.272 |
0.143 |
1.9 |
272.246
|
Sequence luc
Sent luc PCR products and sheared gDNA for sequencing with four primers
JW miniprepped other gRNAs for me
Purified nested PCR of siRNA samples and set up 15ul annealing reaction for surveyor assays. annealed with std protocol in thermocycler
| cell
|
drug
|
gRNA
|
rep
|
260
|
280
|
260/280
|
ng/µL
|
ul for 400ng
|
water to 13.5
|
| Gal4-EED |
+dox |
g034 |
1 |
0.139 |
0.073 |
1.906 |
139.498 |
2.87 |
10.63
|
| Gal4-EED |
+dox |
g034 |
2 |
0.139 |
0.074 |
1.879 |
138.725 |
2.88 |
10.62
|
| Gal4-EED |
+dox |
g034 |
3 |
0.122 |
0.064 |
1.908 |
122.328 |
3.27 |
10.23
|
| Gal4-EED |
+dox |
g048 |
1 |
0.062 |
0.034 |
1.818 |
61.807 |
6.47 |
7.03
|
| Gal4-EED |
+dox |
g048 |
2 |
0.071 |
0.039 |
1.837 |
71.353 |
5.61 |
7.89
|
| Gal4-EED |
+dox |
g048 |
3 |
0.068 |
0.039 |
1.761 |
68.083 |
5.88 |
7.62
|
| Gal4-EED |
+puro |
g034 |
1 |
0.111 |
0.059 |
1.9 |
111.251 |
3.60 |
9.90
|
| Gal4-EED |
+puro |
g034 |
2 |
0.158 |
0.085 |
1.864 |
157.69 |
2.54 |
10.96
|
| Gal4-EED |
+puro |
g034 |
3 |
0.139 |
0.074 |
1.881 |
139.166 |
2.87 |
10.63
|
| Gal4-EED |
+puro |
g048 |
1 |
0.067 |
0.037 |
1.803 |
67.196 |
5.95 |
7.55
|
| Gal4-EED |
+puro |
g048 |
2 |
0.081 |
0.044 |
1.851 |
81.19 |
4.93 |
8.57
|
| Gal4-EED |
+puro |
g048 |
3 |
0.07 |
0.04 |
1.76 |
69.58 |
5.75 |
7.75
|
Purified mCh stop donor PCR reactions
| Donor
|
260
|
280
|
260/280
|
ng/µL
|
| Stop 1 |
0.141 |
0.076 |
1.87 |
141.309
|
| Stop 2 |
0.122 |
0.065 |
1.884 |
122.473
|
| Stop 3 |
0.101 |
0.055 |
1.837 |
100.758
|
Cut first 9 samples with 1ul surveyor and 1ul enhancer for 1hour at 42deg. negative controls for each sample, added 2ul water. added 2ul stop solution after incubation, diluted 1:20, ran on bioanalyzer.
| cell
|
drug
|
gRNA
|
rep
|
| Gal4-EED |
+dox |
g034 |
1
|
| Gal4-EED |
+dox |
g034 |
2
|
| Gal4-EED |
+dox |
g034 |
3
|
| Gal4-EED |
+dox |
g048 |
1
|
| Gal4-EED |
+dox |
g048 |
2
|
| Gal4-EED |
+dox |
g048 |
3
|
| Gal4-EED |
+puro |
g034 |
1
|
| Gal4-EED |
+puro |
g034 |
2
|
| Gal4-EED |
+puro |
g034 |
3
|
Results
background smearing and high variability. think maybe i need new surveyor and/or enhancer. this batch was ordered in january
Transfected KAH126 cells in 6-well plate with 3
realized if i make two double strand breaks, i'll get a large deletion. just used either the top of bottom dsb plasmid with the donor for stop 1 or stop 2
| '
|
ug plasmid
|
ug donor
|
| 3T |
1 |
0
|
| 3T d1 |
0.5 |
0.5
|
| 3T d2 |
0.5 |
0.5
|
| 3B |
1 |
0
|
| 3B d1 |
0.5 |
0.5
|
| 3B d2 |
0.5 |
0.5
|
Froze down KAH154s treated with Cas9 from last CSH course. Two vials per treatment type.
added new media to T75 flask of gal4-eeds+puro. should freeze them tomorrow.
| 
Project name
