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

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

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 mix 7.5 435
750nmol F/R primer mix 3 174
PCR water 2.5 145
total 13 754


Primer set 3 master mix

' 1 well 33
2x SYBR mix 7.5 247.5
750nmol F/R primer mix 3 99
PCR water 2.5 82.5
total 13 429


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
Luc14 34 1 0.069 0.037 1.855 69.338 5.77 1.50 7.73
Luc14 34 2 0.064 0.035 1.848 64.247 6.23 1.50 7.27
Luc14 34 3 0.127 0.068 1.866 126.603 3.16 1.50 10.34
Luc14 KAH228 34 1 0.051 0.028 1.815 51.075 7.83 1.50 5.67
Luc14 KAH228 34 2 0.034 0.019 1.764 33.857 11.81 1.50 1.69
Luc14 KAH228 34 3 0.03 0.016 1.846 29.947 13.36 1.50 0.14
Gal4+dox KAH228 34 1 0.043 0.024 1.831 43.229 9.25 1.50 4.25
Gal4+dox KAH228 34 2 0.053 0.029 1.815 52.832 7.57 1.50 5.93
Gal4+dox KAH228 34 3 0.031 0.016 1.885 30.769 13.00 1.50 0.50




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
Luc14 54 1 0.199 0.106 1.876 159.233 2.51 1.50 10.99
Luc14 54 2 0.189 0.1 1.879 150.896 2.65 1.50 10.85
Luc14 54 3 0.181 0.096 1.873 144.402 2.77 1.50 10.73



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