Jane,
Gaetan Burgio
1

Hi Gaetan,

I agree that more experiments need to be done before we can conclude what happened here. But I have two more points to make and I wish they could be a little bit helpful for your experiments. 1) Your answer reminds me that it is normal that your PCR product sequences match perfectly with your primer but not with the Genomic sequences. Remember that when we do cloning, we always modify the primer at 5’end to introduce restriction-cutting sites? As long as the 3’end of the primer matches pretty well with the target sequence (It is the mouse genomic sequences here), you will be able to amplify the targets with modified 5’ end sequences. The PCR product sequence should always fully match with PCR primers even it is unspecific amplification, if I understand the PCR principle correctly. 2) I think your major purpose here is to test if NgAgo could edit genome in vivo in body temperature (or whatever temperature you used for your cell culture). To make it easy, I think you should get rid of everything (including NgAgo, 5’P Oligo) except for the Genomic DNA before the PCR. As you mentioned, you could use phenol to denature all the proteins. If you suspect that NgAgo has other enzymatic activities at higher temperatures, you may design separate in vitro experiment to confirm that. That’s all I can think of for now. It’s also a great pleasure to discuss with you. Good luck with your experiments!

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