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

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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.