How can you be sure that you're digesting in the right locations if you don't know the location of your insertion site? It seems like it could provide 90% accurate data, but the chance of losing something due to fragment sizes being too large or small is there.
Maybe you can put me at ease.
-SG
On Thu, Jul 12, 2018 at 9:58 AM, Scott <synbiofablab@gmail.com> wrote:
Splinkerette pcr is a nice protocol. Haven't tried it myself. I disagree on your assertion that inverse PCR is difficult. I've used it numerous times to identify mouse transgene insertion sites as well as some bacterial transposon integrations. No mechanical shearing needed, only restriction digest. You have to pick the right enzyme(s) depending on your transgene. It has always worked well in my hands.--
On Thursday, July 12, 2018 at 9:44:14 AM UTC-7, Skyler Gordon wrote:Inverse PCR is also going to be difficult unless you have some sort of mechanical DNA shearing method. Common enzymatic and chemical shearing will break the DNA down into pieces that are just too small.I would advise using a splinkerette pcr, or following the protocol that I've attached. Both are simple, rather elegant ways to ensure your PCR products come from the desired location in your genome.-SG
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