Anything and everything cloning: Go...
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Thank you for your help. I knew the plasmid portion of the primers might be problematic. I am trying to clone these genes in the exact same position in the plasmid as a previous post-doc did with a different variant of the gene. She used NdeI/BamHI sites for insertion, but unfortunately, the Bovis variants have NdeI sites in the middle of the gene. I will take a look at the alternative site that you recommended and see if I can make it work for our purposes. I guess I could always try to inverse PCR a different restriction site instead of the NdeI site, but your method looked so appealing. I hope that I can get it to work at some point!
As far as the GC content in the plasmid-primer portion - at what Tm should I stop considering the primer as compatible? 70C? These genes are all surrounded by GC-rich areas upstream, and GC-poor areas downstream, so the GC content difference also appears to be a problem for designing primers.
Hi, so I was just introduced to this technique a couple of weeks ago, and I am having no luck getting it to work. I have sequenced my plasmid and my desired inserts and everything is as expected. I have made the primers according to this website and I am able to amplify the genomic DNA with those primers to get my desired insert product. I have tried both the 2-step and the 3-step secondary PCR using the extension times recommended by this website. After DpnI digest (I have done both 3 hrs and O/N), I electroporate them into a DH10B strain, recover in LB and then plate on selective media. When I do colony PCR the next day using the vector primers (T7 Promoter/T7 Terminator), 20+ colonies, all of them are negative for insert. Instead, all of the colonies carry the original plasmid sequence. I have also digested the same amount of plasmid with DpnI without running PCR on it, and I get no colonies, as expected. All of these attempts have used the same batch of electrocomp. cells. Is it possible that I am somehow amplifying the plasmid? Any help would be appreciated! I would love to get this method up and running in our lab.
Thanks!
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