Does a T7 endonuclease assay always work?
Currently I'm using a plasmid pSpCas9(BB)-2A-Puro (PX459) on HEKs to test my guides. Prior I sequence to check guides have been successfully cloned in etc. Following seven days of puromycin selection I ran the T7 and only got a single band on my gel (first and only time).
Primer design has been good regarding Tm, specificity etc., and the T7 is for a 2000bp fragment with the cut expected to be at around 600 bp upstream of my 5' primer. I'm performing PCR clean-up etc.
On talking to others, some suggest that the T7 is not reliable if you have low efficiency and to use other methods. I'm open, but I want to make sure that I am doing all I can to make sure that its a problem with me doing the assay and not necessarily 'bad' guides.
Any suggestions/ comments/ feedback much appreciated!
Many thanks,
Cengiz
In my case,
We are not PCR clean-up sample since in this step you will lose so much PCR product. And if the efficiency is not that high you will not see the cut bands.
You can check near the cut side if there is a restriction endonuclease side. This way you can definetely understand wheither your DNA has been cut and end-joined or not.
Hi Cengiz Akbulut ,
If the targeted editing efficacy is not high enough, it should be difficult to see the edited bands using T7 method.
I suggest you design primers to amplify small PCR products then use polyacrylamide gel running follow the paper: " An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system"
Best
Hi,
Thank you for your answers and input.
I ran everything again and set up a couple extra-internal controls and it worked pretty well second time round.
- I used a PCR clean-up as it was suggested any residual enzymes might inhibit endonuclease activity etc.
- I was thinking that I could design small primer fragments for the next time I carry out this assay - should it be looking for a different CRISPR cut.
I wonder now, that if you have loaded equal amount of DNA into wells, does the intensity of lower bands indicate cutting efficiency of the guides. But think this is very crude assumption and potentially other methods should be explored for cutting efficiency.
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