Haynes Lab:Notebook/CRISPR Editing/2015/02/03: Difference between revisions

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==02/03/2015==
==02/03/2015==
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I added enzymes to my insert sample (which was PCR-purified the day before) for digestion. I first added water to a ?μL tube, then I added the sample, buffer, and two enzymes.  
I added enzymes to my insert sample (which was PCR-purified the day before) for digestion. I first added water to a 200μL PCR tube, then I added the sample, buffer, and two enzymes.  
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Revision as of 13:21, 6 February 2015

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02/03/2015

I added enzymes to my insert sample (which was PCR-purified the day before) for digestion. I first added water to a 200μL PCR tube, then I added the sample, buffer, and two enzymes.
Restriction digest

Thing vol in ul
1ug sample 9.17
H2O 6.83
10x buffer 2
PstI 1
BbsI 1
Total 20


Purified with PCR clean up kit

Sample Read# 260 280 260/280 ng/µL
insert for ryan 0.012 0.007 1.62 12.1



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


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