Haynes Lab:Notebook/CRISPR Editing/2016/06/01

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06/01/2016

Plate 5
L 25 F/I set 3
G 25 F set 3
L/G 31 F/I/input pset 2

Primer set 2 master mix

' 1 well 58
2x SYBR mix7.5435
750nmol F/R primer mix3174
PCR water2.5145
total13754


Primer set 3 master mix

' 1 well 33
2x SYBR mix7.5247.5
750nmol F/R primer mix399
PCR water2.582.5
total13429


Image:16.06.01 qPCR Plate 5 screen shot.png



Set up annealing reaction for below purified nested PCR reactions:

Cell type treatment gRNA rep 260 280 260/280 ng/µL ul for 400ng ul ann buff ul H2O
Luc143410.0690.0371.85569.3385.771.507.73
Luc143420.0640.0351.84864.2476.231.507.27
Luc143430.1270.0681.866126.6033.161.5010.34
Luc14KAH2283410.0510.0281.81551.0757.831.505.67
Luc14KAH2283420.0340.0191.76433.85711.811.501.69
Luc14KAH2283430.030.0161.84629.94713.361.500.14
Gal4+doxKAH2283410.0430.0241.83143.2299.251.504.25
Gal4+doxKAH2283420.0530.0291.81552.8327.571.505.93
Gal4+doxKAH2283430.0310.0161.88530.76913.001.500.50




Checked DNA concentrations of Luc14, Luc14 KAH228, Gal4 KAH228 g034 nPCR from yesterday. Looks great, set up annealing reaction.

Surveyor of Luc14 g034 and g044 samples

Surveyor of Luc14 KAH228, Gal4 KAH228 g034 samples



Nested PCR of Luc14 g054 with P149/162
Ran on gel, single band, no band in -ctrl lane. Column purified, set up for annealing reaction.

Cell type gRNA rep 260 280 260/280 ng/µL ul for 400 ul ann buff ul water
Luc145410.1990.1061.876159.2332.511.5010.99
Luc145420.1890.11.879150.8962.651.5010.85
Luc145430.1810.0961.873144.4022.771.5010.73



P197/198 PCR of g055 rep 2. Hadn't been working so I tried different concentrations of genomic DNA and 1ul of Phusion.

5ul gDNA, worked!
10ul gDNA, didn't work
15ul gDNA, didn't work
20ul gDNA, didn't work
neg ctrl, no band


Nested PCR of g055, P153/160




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