Hey, I am facing the a problem in ligation transformation. my clones for both 1:3 and 1:6 molar ratio ligation mix giving false positives, I am not understanding what might be the problem for this. I believe digestion is complete for both plasmid and insert. ligation has worked well as I can see colonies for the single digested vector that should give colonies(positive control). What is the problem I am not able to understand. In my colony PCR I have got positive result with the clone but when doing plasmid isolation by alkaline lysis method, I am unable to even get DNA on the gel picture. When doing plasmid isolation in the solution 2 step I can see some precipitate kind of thing that too only for few clones and these clones fail to give any DNA after isolation. The lane in the gel picture gives blank image with no DNA. What can be the possible reason for this? Any suggestions?
How do you proceed for the plasmid miniprep ? Did you send your PCR products to be sequenced ? Which type of vector are you using ?
Hi Shifa!
If I understand your question correctly, you have positive PCR results with DNA isolated from clones (transformed with a plasmid with gene of interest) but unable to get desired plasmid/no plasmid
Please check the following.
1. Are your PCR primers specific to your gene of interest and don't amplify your host gDNA (is the same gene present in the host or any highly similar sequences present).
2. Include plasmid-specific primers to check for transformation. (You can design a primer pair that amplifies part of your gene and plasmid.
3. To avoid false positives in transformations, you can increase antibiotic concentration.
Cheers!!
Thanks
Pranay
I agree with Jena-Pierre Dangy. It seems that there is something wrong with the minipreps. Normally, if you grow the cells in the presence of the proper antibiotic, you should at least get the plasmid, regardless whether it contains the correct insert. Do you use a miniprep kit or do you just perform an alkaline lysis preparation according to Binrboim and Doly ?
I use appropriate antibiotic with specified concentration. The culture is growing well and I use Midiprep alkaline lysis method to isolate plasmid. So no problem with the growth of the cells in antibiotic media. The DNA is seen for few clones with no shift and in few clones no DNA is seen that is the problem. Why this difference when all the clones are screened in the same way without any change in there culturing or isolation or anything else is my question?.
Hard to follow what you have really done. Do you load the intact plasmid DNA or are you doing a restiction cuting of your plasmid ? One explanation might be that some of your clones are not really recombinant. Very hard to give you any recommandation with too few details on your experiment set-up
Try for restriction digestion to check the presence of insert rather than colony PCR screening.
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