Haynes Lab:Notebook/CRISPR Editing/2015/02/03

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02/03/2015

Digested 400ng of purified PCR products from yesterday. Also duplicated with no enzyme, added 1ul elution solution:

Sample Concentration ul for 400ng ul Elut sol'n ul buffer 4 ul PfoI
Luc14 g034 1 29.05 13.77 3.23 2 1
Luc14 g034 2 28.85 13.87 3.13 2 1
Luc14 g034 3 39.05 10.24 6.76 2 1
GAL4-EED +dox g034 1 38.92 10.28 6.72 2 1
GAL4-EED +dox g034 2 37.78 10.59 6.41 2 1
GAL4-EED +dox g034 3 42.04 9.51 7.49 2 1

Let reaction go for 3 hours, diluted 1:10, ran on bioanalyzer
Hybridization for Surveyor, 400ng in 20ul reactions, 2 reactions each (for +/- nuc):

Sample Concentration ul for 400ng ul elut sol'n ul 10x hybridization buff
Luc14 g034 1 29.05 13.77 4.23 2
Luc14 g034 2 28.85 13.87 4.13 2
Luc14 g034 3 39.05 10.24 7.76 2
GAL4-EED +dox g034 1 38.92 10.28 7.72 2
GAL4-EED +dox g034 2 37.78 10.59 7.41 2
GAL4-EED +dox g034 3 42.04 9.51 8.49 2


Added 1ul surveyor nuclease, 1ul enhancer (made a MM). Added 2ul elution solution to -nuc samples.
Incubated at 42°C for 45 minutes.
Add 2ul stop solution to every tube (even -nuc).
make 1:10 dilution, analyze on bioanalyzer