05/16/2015
Cloning GFP behind Cas9
Will do ligation before and after purifying with Qiaquick PCR purification kit which will get rid of the 23 basepairs cut out of the backbone and the >10bps cut off the ends of the insert
before purification
- 50ng backbone (8486bp) = 50ng /(1000ng/50ul) = 2.5ul
- 10ng insert (824bp) = 10ng / (40ng/25ul) = 6.25ul
mixed backbone and insert, backbone only, brought both to 10ul with water
added 10ul 2x Roche ligation buffer
added 1ul Roche T4 ligase
mixed and spun
incubated at RT on benchtop for 15-20mins
moved 2ul of each reaction to a new tube, added 10ul water
water only control
added 50ul of my DH5αTs or 50ul of KAH's DH5αTs
incubate on ice for 30min
heat shock at 42°C for 35s
incubate on ice for 2min
add 750ul SOC to each tube
shake at 37°C for 1hour
plate all cells
Cell type
|
plasmid 1
|
plasmid 2
|
rep
|
260
|
280
|
260/280
|
ng/µL
|
ul for 400ng
|
Luc14 |
GFP |
g034 |
2 |
0.033 |
0.019 |
1.791 |
33.354 |
11.99256461
|
Luc14 |
GFP |
g034 |
3 |
0.027 |
0.014 |
1.953 |
26.814 |
14.91758037
|
Luc14 |
GFP |
KAH228 |
2 |
0.03 |
0.016 |
1.853 |
30.427 |
13.14621882
|
Luc14 |
GFP |
KAH228 |
3 |
0.033 |
0.016 |
2.096 |
32.56 |
12.28501229
|
Luc14 |
KAH228 |
g034 |
2 |
0.032 |
0.018 |
1.814 |
31.868 |
12.55177608
|
Luc14 |
KAH228 |
g034 |
3 |
0.032 |
0.017 |
1.869 |
31.64 |
12.64222503
|
Gal4-EED +dox |
GFP |
g034 |
2 |
0.033 |
0.017 |
1.916 |
33.466 |
11.95242933
|
Gal4-EED +dox |
GFP |
g034 |
3 |
0.027 |
0.014 |
1.953 |
26.521 |
15.08238754
|
Gal4-EED +dox |
GFP |
KAH228 |
2 |
0.03 |
0.017 |
1.75 |
30.466 |
13.12939014
|
Gal4-EED +dox |
GFP |
KAH228 |
3 |
0.023 |
0.012 |
1.964 |
22.794 |
17.54847767
|
Gal4-EED +dox |
KAH228 |
g034 |
2 |
0.025 |
0.013 |
1.935 |
24.575 |
16.27670397
|
Gal4-EED +dox |
KAH228 |
g034 |
3 |
0.029 |
0.015 |
1.925 |
29.369 |
13.61980319
|
cell type
|
ug plasmid
|
ul lipo
|
rep
|
260
|
280
|
260/280
|
ng/µL
|
ul for 400ng
|
Gal4-EED |
0.5 |
3 |
1 |
0.034 |
0.018 |
1.91 |
33.545 |
11.92428082
|
Gal4-EED |
0.5 |
3 |
2 |
0.031 |
0.017 |
1.886 |
31.165 |
12.83491096
|
Gal4-EED |
1 |
3 |
1 |
0.03 |
0.015 |
1.939 |
29.994 |
13.33600053
|
Gal4-EED |
1 |
3 |
2 |
0.031 |
0.018 |
1.721 |
30.674 |
13.04035991
|
Gal4-EED |
0.5 |
5 |
1 |
0.028 |
0.015 |
1.943 |
28.44 |
14.06469761
|
Gal4-EED |
0.5 |
5 |
2 |
0.027 |
0.014 |
1.921 |
27.34 |
14.63057791
|
Gal4-EED |
1 |
5 |
1 |
0.03 |
0.016 |
1.884 |
30.303 |
13.2000132
|
Gal4-EED |
1 |
5 |
2 |
0.029 |
0.015 |
1.938 |
28.813 |
13.88262243
|
'
|
Cell type
|
plasmid 1
|
plasmid 2
|
rep
|
ng/µL
|
ul for 400ng
|
ul annealing buff
|
ul water
|
sur
|
1 |
Luc14 |
GFP |
g034 |
2 |
33.354 |
11.99 |
2.00 |
6.01 |
+
|
2 |
Luc14 |
GFP |
g034 |
3 |
26.814 |
14.92 |
2.00 |
3.08 |
+
|
3 |
Luc14 |
KAH228 |
g034 |
2 |
31.868 |
12.55 |
2.00 |
5.45 |
+
|
4 |
Luc14 |
KAH228 |
g034 |
3 |
31.64 |
12.64 |
2.00 |
5.36 |
+
|
5 |
Gal4-EED +dox |
GFP |
g034 |
2 |
33.466 |
11.95 |
2.00 |
6.05 |
+
|
6 |
Gal4-EED +dox |
GFP |
g034 |
3 |
26.521 |
15.08 |
2.00 |
2.92 |
+
|
7 |
Gal4-EED +dox |
KAH228 |
g034 |
2 |
24.575 |
16.28 |
2.00 |
1.72 |
+
|
8 |
Gal4-EED +dox |
KAH228 |
g034 |
3 |
29.369 |
13.62 |
2.00 |
4.38 |
+
|
9 |
Luc14 |
GFP |
KAH228 |
2 |
30.427 |
13.15 |
2.00 |
4.85 |
+
|
10 |
Luc14 |
KAH228 |
g034 |
2 |
31.868 |
12.55 |
2.00 |
5.45 |
-
|
11 |
Gal4-EED +dox |
KAH228 |
g034 |
2 |
24.575 |
16.28 |
2.00 |
1.72 |
-
|
|