Haynes Lab:Notebook/CRISPR Editing/2014/11/25

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Gel of Surveyor PCR yesterday showed multiple bands. Ran 30μL of those PCR reactions on a gel (largest combs) and cut out the top band. It looked like maybe there were two bands really close to each other but you can't see that in the picture. The bottom band was smiley and dipped a little below 1500bp. Cut both bands out. Added 300μL of a new bottle of Zymo ADB buffer and incubated at 55°C until the gel slices dissolved. Loaded onto a Sigma miniprep column, spun for 1 min at full speed. Washed twice with 500μL of Sigma PCR clean-up kit wash buffer. Eluted in 30μL elution solution.

Re-ran the PCR from yesterday, exactly the same, just did 30 cycles instead of 36. Ran 5μL of the first PCR reaction of each one on a gel, single band (fuzzy bands at the bottom will go away after purification, don't match the length of the off target bands from the previous run): [insert gel image] Used Sigma PCR cleanup kit. Eluted in 50μL sigma elution solution

Sample Read# 260 280 260/280 ng/µL
Luc14 gel ext 0.063 0.033 1.926 63.311
G029 gel ext 0.04 0.021 1.938 39.756
Luc14 col. pur. 0.078 0.041 1.883 78.095
g029 col. pur. 0.083 0.044 1.905 83.455

Used the second PCR run (column purified) for Surveyor. Annealed with the protocol from the assay:

Sample 400 ng ul Elution sol ul Hyb buff
Luc14 gDNA +nuc 5.12 3.88 1
Luc14 gDNA +nuc 5.12 3.88 1
g029 Luc14 +nuc 4.82 4.12 1
g029 Luc14 +nuc 4.82 4.12 1

Added 1ul Enhancer and 1 ul Surveyor nuclease to the +nuc samples and 2ul elution solution to the -nuc samples. Incubated at 42 for 45min in PCR machine.
Added 1.32ul stop solution to all the samples, brought to Biodesign for Bioanalyzer