Haynes Lab:Notebook/CRISPR Editing/2015/01/09

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

01/09/2015

Sample Read# 260 280 260/280 ng/µL
31 0.114 0.061 1.854 113.641
35 0.097 0.051 1.913 97.073
38 0.117 0.062 1.887 117.472
40 0.103 0.055 1.89 103.387
41 0.128 0.067 1.907 128.36
42 0.102 0.055 1.855 101.652
48 0.109 0.06 1.806 108.503
49 0.096 0.051 1.871 95.959


Sample Read# ng/µL ul for 400ng ulH2O ul ann buff
31 113.641 3.52 9.98 1.5
35 97.073 4.12 9.38 1.5
38 117.472 3.41 10.09 1.5
40 103.387 3.87 9.63 1.5
41 128.36 3.12 10.38 1.5
42 101.652 3.93 9.57 1.5
48 108.503 3.69 9.81 1.5
49 95.959 4.17 9.33 1.5



Had 2-15ul annealing reactions for all 22 gRNA treated Luc14 samples. Added 1ul Surveyor enzyme and 1ul of enhancer (mixed together first) to each +nuc tube and 2ul of elution solution to the -nuc tubes. Incubated at 42degC for 45 minutes, added 1.7ul stop solution to every tube.

Analysis with bioanalyzer
Made 1:20 dilutions of each Surveyor reaction. Ran one ul on each chip. Ran untreated samples +/-Luc on every chip. Data looks really solid, can select some gRNAs to proceed with.

Analysis with 2% agarose gel
Added 3.74ul 6x loading dye to the remaining 17.7ul of surveyor reaction. Ran on a large 2% agarose gel. Made the gel very thick so I could fit all the reaction in the well but some still spilled out. I also took a long time to load the gel so I think that messed up some of the early lanes. It looks like there are two bands in those lanes, even in the ladder. I think maybe I ran it too fast (100V). I need to learn more about 2% gels. The bands are consistent with the bioanalyzer data but it's not close to publishable data.



Thawed Luc14 gal4-eed cells for experiments