I have been trying to have a good AAV titration for my production and I have tried different plasmids to set up as a standard curve but each one behaves differently. i am trying to use ITR for quantification. Any suggestion would be great.
Hi Khaled
I use this protocol from Addgene which works pretty well.
https://www.addgene.org/protocols/aav-titration-qpcr-using-sybr-green-technology/
You can use it with pretty much any plasmid containing an ITR. Personally I use a plasmid with single ITR as a standard (pX458, CRISPR plasmid from Addgene). Plasmids with two ITRs could theoretically produce more noise, depending on how close together they are.
Best
Hi Khaled, it worked pretty well with my plasmid - I haven't tried their recommended plasmid.
If you have problems with the protocol, another option might be to try lysing the particles first. The denaturation step should do this, but I've seen some protocols where they do a lysis step initially before qPCR.
Do you start with 2*109 for 10x serial dilution or do you start with 10*109 For your standard plasmid
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