Haynes Lab:Notebook/CRISPR Editing/2015/07/20

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

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 T0.2580.1331.945258.246
gn001 B0.30.1561.919299.748
gn003 T0.2950.1531.93294.921
gn003 B0.2560.1341.918256.118
g0050.2690.141.917269.101
g00130.2090.111.891208.912
g0570.2260.1181.916225.742
g0580.2420.1261.913241.86
g0590.2050.1071.916205.376
g0600.2720.1431.9272.246



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+doxg03410.1390.0731.906139.4982.8710.63
Gal4-EED+doxg03420.1390.0741.879138.7252.8810.62
Gal4-EED+doxg03430.1220.0641.908122.3283.2710.23
Gal4-EED+doxg04810.0620.0341.81861.8076.477.03
Gal4-EED+doxg04820.0710.0391.83771.3535.617.89
Gal4-EED+doxg04830.0680.0391.76168.0835.887.62
Gal4-EED+purog03410.1110.0591.9111.2513.609.90
Gal4-EED+purog03420.1580.0851.864157.692.5410.96
Gal4-EED+purog03430.1390.0741.881139.1662.8710.63
Gal4-EED+purog04810.0670.0371.80367.1965.957.55
Gal4-EED+purog04820.0810.0441.85181.194.938.57
Gal4-EED+purog04830.070.041.7669.585.757.75



Purified mCh stop donor PCR reactions

Donor 260 280 260/280 ng/µL
Stop 10.1410.0761.87141.309
Stop 20.1220.0651.884122.473
Stop 30.1010.0551.837100.758



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+doxg0341
Gal4-EED+doxg0342
Gal4-EED+doxg0343
Gal4-EED+doxg0481
Gal4-EED+doxg0482
Gal4-EED+doxg0483
Gal4-EED+purog0341
Gal4-EED+purog0342
Gal4-EED+purog0343


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
3T10
3T d10.50.5
3T d20.50.5
3B 10
3B d10.50.5
3B d20.50.5



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.



Personal tools