I have performed cloning for the gene of interest using pcrII TOPO vector. After the slection steps and innoculation steps I isolated the plasmids using QIAGEN kit and then measured using nanodrop. Then I performed PCR from a fraction of the isolated plasmid to check if the gene of interest has been incorporated within the plasmid and the PCR worked. I sent the samples for sequencing and we have used M13 reverse primer. But what we dont know if our products are fwd oriented or reverse oriented. I have been asked to do sequence alignment using ebi.ac.uk but I dont understand how to proceed and what I infer. I would appreciate if anyone can tell me how to go about or proceed.
If you know the gene sequence, you can make one primer from the gene and one primer from the backbone for PCR to figure out the orientation of the inserted gene. Beside, you should see a 'start codon' and a 'stop codon' for the gene.
Hi Ratish, this is a common problem when doing non directional cloning. It can be solved by 1) analysis with restriction enzymes 2) PCR or 3) sequencing. In either case, it is really helpful to get the map and the sequence of the plasmid (internet, supplier catalogues, etc..) and at least some indication about sequences present in your PCR product. You have the sequences of the primers you used for PCR. Since they are at the ends of your PCR product, try to find these in your sequencing data.
I agree with Simon, it definitely helps to know how the ends of the of your products look which might to help you find the correct order in the sequence. Unfortunately a lot of restriction sites are palondromic so that's also something to think about.
If you know the gene sequence, you can make one primer from the gene and one primer from the backbone for PCR to figure out the orientation of the inserted gene. Beside, you should see a 'start codon' and a 'stop codon' for the gene.
First study the vector map carefully.
Try to find forward/ reverse primers In your sequence data generated using M13 reverse primer. If you find forward primer sequence (without revere complementing it) it is in forward orientation with SP6 promoter. If you find reverse primer used for PCR (without reverse complementing it) it is in reverse orientation.
To verify further, In case of forward orientation with SP6 primer, your forward primer is nearer to BstXi site than to EcoRV site. In the other orientation your reverse primer is nearer to BstXi site than to EcoRV site
Hope it is useful
- Badges
- Science method