PCR of plasmid miniprep from Agrobacterium - Can't get PCR product out of plasmid miniprep (Apr/23/2005 )
Hi, I transformed agrobac with a certain Ti plasmid containing my insert. Then, I miniprepped the agrobac using Qiagen's alkaline lysis procedure and did PCR (Novagen's KOD HotStart) on the miniprep (primers for my insert). But I don't see any bands for PCR products on the gel. I'm assuming I can't see the plasmid itself which I miniprepped, because it has a low copy number in agrobac. Anyone ever succeeded using Qiagen's miniprep on agro and PCR-ed the plasmid or know what could be wrong? If you have any other suggestions for doing miniprep and PCR on agrobac that would help as well. Thanks alot.
In my experience, i wasn't also able to see my plasmid in the gel but when i ran it through pcr i was able to get my expected amplicon and i think your plasmid is a lot more clean than mine coz i used the common alkaline lysis method, so if eveything's ok with your primer, pcr cocktail, etc.. the problem might be in your plasmid. I assumed that your culture was grown in a medium with antibiotic, so you may first try to amplify your antibiotic marker gene. if you're abe to get an amplicon proceed on checking again your gene of interest. If you're sure that your pcr profile, cocktail, etc.. are ok but still no product, then you might just have the marker in your insert but without your gene of interest. Another alternative is to do pcr using a lysate of your agrobact. When i do pcr of the plasmid, i usually include a lysate or i picked a colony of the agro and use them as control. hope this helps.