T7 endonuclease assay for KO clone: searching for protocol of direct PCR.

I am doing a KO RNP approach on suspension cell lines. I will be doing a limiting dilution to isolate single clones. My plan is to have 3 96-well plates plated with 0.5 cells/well, any comments?

I will then be doing a T7 endonuclease: I am having trouble getting the PCR product. From column purified genomic DNA, I am able to get the PCR product flanking the cut site. But direct PCR is not working.

My direct PCR protocol is a simple 1x10E3-1x10E5 cells resuspended in tris buffer and boiled for 10 mins, and using that for the PCR reaction.

Any robust and simple direct PCR protocol you recommend?

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Seashell Lichover 3 years ago

you should look at doing a TIDE analysis instead - much simpler and more informative!

Blue Harpyover 3 years ago

For direct PCR from small amount of cells I usually:

1) Dissociate a 96 well with 50 uL trypsin, TrypLE, or other dissociating agent

2) After cells are broken up, remove 10% (5 uL) to a PCR plate, replate the remaining 90% of cells on a 96 or 48 well plate

3) Add 5 uL of a proteinase K mix (10 ug Proteinase K, 4 uL TE per reaction) to the PCR plate

4) Incubate in a thermocycler at 50 C for 45 minutes, then 95 C for 10 minutes (to denature ProtK)

5) Use lysate directly as a PCR template (I usually use 2 uL per 25 uL rxn)

This has worked well for me even with small cell numbers.

Hope this helps

Brown Lamiaover 3 years ago

Jack - Are you trying to screen your KO clones by surveyor? That is a pretty low throughput way to screen clones. I would recommend western blot of the clones or a quick PCR based genotyping assay for doing this. Better yet, if there is some phenotype you can rapidly screen that's even better.

https://www.lucigen.com/QuickExtract-DNA-Extraction-Solution/

That is a reagent you can use for quickly preparing gDNA for PCR and subsequent surveyor. Make sure that for surveyor you have a very clean PCR product and that you do a clean up prior to using the T7. I also recommend doing a 1-2 hour incubation with T7 - the shorter incubation times usually result in incomplete cutting, which makes the data hard to interpret.

Gold Gremlinover 3 years ago

Thanks for the tips regarding surveyor. I ended up confirming KO by western blot because my surveyor results were strange. Haven't had the best luck with it. Going to try TIDE next for something to do in between waiting for enough cells for western.

Sienna Selkieover 3 years ago

the direct PCR protocol worked very well.

I am looking into TIDE, but it seems it relies on sequencing, which might not be cost-effective. Can you elaborate on the approach or share a protocol.

I am trying to avoid western; can you elaborate on the "quick PCR genotyping". My protein to KO is an intercellular NOD like receptor; so no phenotypes to select.

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