Hello
I used crispr cas9 system to knock out gene.
I transfected vector (SBI) to EPC cell line and selected cell
and extract gDNA from 1x10^6 cells/ml
and did PCR about 1000bp size that targeted region located in the middle of the
PCR template.
and I load all of the PCR product and get right band so I gel-ex the band.
to confirm the muation I did t7e1 (NEB) assay follow manual.
at the manual the dna concentration is 200ng
but isn't it too low concentration?
the band is not clear so I did 1000ng of dna
at that time negative control (untransfected epc cell )
also cutted in two.
(I did nested pcr also)
I used Agarose gel and TAE Buf
some people said used polyacrylamide tbe gel
is it make big different?
Hello
I'm having same problem. Were you able to boost T7E1 assay efficiency?
In my case, after several try on agarose gel, I load the sample on acrylamide gel and detect the band. so how about try on acrylamide gel? :) or just do a Cloning and do a Sanger sequencing is more easy way to know the working of crispr
T7EI assay worked for me when I used 1000ng instead of 200ng for some targets. I used it using genomic DNA from transformed plant protoplast cells . For some targets it worked fine and others not. It also depends on transformation efficiency. If you have low rates of transformation, the bands are too faint.
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