Hello,
I'm currently trying to delete a 5kb gene from my plasmid, using the Directed-site mutagenesis kit from neb but the PCR part is not working and I don't know why. I designed the primers with https://nebasechanger.neb.com/ and followed exactly the protocol but I obtained nothing on my agarose gel in the end.
Could you please help me find out why it is not working?
Thank you
Dear Sam
Did you load the Plasmid PCR in the gel before dpnI digestion and bacterial trasfromation? How was the band?
Can you share the sequence of primers and template?
Did you deleted a ORF cloned into the plasmid or a part of the plasmid? In the last case, which was the function of this gene?
best regards
Manuele
Hi Manuele,
Thank you for your quick answer.
I loaded 5uL on the gel before the treatment with DpnI and I didn't get any band. I'm used to this kit and never had any trouble before, but I never tried to delete a 5kb ORF in a 12kb plasmid though.
Neb says that they managed to do it with 20kb plasmids so it should be possible.
I downloaded a word file with the ORF i want to delete and the primers I used.
The ORF was cloned in the plasmid by another lab and we just want to replace it with another gene to express a different protein.
Best regards
Sam
some more information would help. For 5kb you would be better off trying an inverse PCR rather than an SDM - presumably in your SDM experiment you expect a primer to bind on either side of a 5kb gene? Not very feasible. Another option would be to introduce RE sites on either side of the gene by SDM.
Gary James Hunter sorry if my question is pretty naive, but I don't understand why it would be "not very feasible"? Indeed I designed 2 primers to amplify the rest of the plasmid without the ORF that I don't want.
It's true that I could introduce RE sites but I wanted to use the SDM cause it's supposed to work and is far less time-consuming. But yes in the end I'll do that I guess.
thank you for your answer.
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