I have to clone a gene in the range of ~3.5 from cDNA and I am having issues designing the primers using software. Basically I want to design the primers that can amplify full-length CDS (start-stop). Even though i input the FL CDS of the gene along with 50bp of UTR's in the primer design softwares, I am not getting the primers that will only amplify CDS completely. Any help please?
Hi , on general when you are using software to chose primers the program is following some rules in terms of size requested, Tm, GC content etc. Me, when I have to chose primers for cloning full length cDNA of a given gene simply I design the primers myself. You can make them a bit longer in order to fit the major rules for primer design. Good luck.
Other possibility is to use In-fusion cloning system from Clontech.
Hi Yordan, Thanks. That is what exactly i did. However when i run PCR with the manually designed primers, i did not see anything. I am wondering now that may be its just the PCR that didn't work. I used EMD Millipore KOD polymerase. Do you have recommendations for PCR?
Hi Upendra, yes could be just the PCR is not working so well. You can try high fidelity DNA polymerase from Clontech. In my experience the choice of well working polymerase is critical. I am using Advantage® HD Polymerase Mix .
Good luck!
Thanks Yordan for your useful tips and particularly In-fusion cloning system sounds interesting. May be i will start amplifying the genomic DNA with these primers to make sure the problem is not with the cDNA (I know it is not but better to be circumspect). One the primers are ok then i will work on the cDNA.
Hi Upendra, did you check the GC% of your primers? Try to keep GC% of forward and reverse primers as close as possible by reducing or increasing the bases.
Hi Shishir, actually i forgot to update the progress here but actually my primers worked with genomic DNA and so it seems the problem is not with primers but with cDNA. My hypothesis for cDNA is that since the gene is very long (~3.5kb), the Super Script IIIRT that i have used to revere transcribe mRNA failed to generate full length transcripts. Since the generated cDNA doesn't have full length transcripts, the primers failed to bind to my gene of interest. Now i am trying a different RT enzyme and hopefully it should fix the problem. Thanks both for your suggestions anyway.
Hope your problem will be solved very soon. I have a question to you. Did you use any specialized PCR kit to amplify your product or normal Taq polymerase with fidelity?
I used Xtreme Hot Start Polymerase enzyme kit for amplification. Normal Taq did not worked.
Thank you very much. We have started a project for cloning a 3.5kb gene. So, your info will be helpful.
Ok good luck with it. I just realized that i haven't given the full name of the kit - it is KOD Xtreme Hot Start Polymerase enzyme kit .
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