My gene of interest is 5.5 kb long one. this gene has been expressed only under stress conditions and I have used NaCl treatment (Salinity stress) to induce the gene expression and I have isolated RNA from the NaCl treated leaves. I have designed primers to amplify the entire 5.5 kb as well as I have designed primers to amplify the N terminal 3 kb and 2.5 kb C- terminal also. I have tried many times but I have not got the full length 5.5 kb gene. Further I have tried to amplify the C- terminal 2.5 kb region alone and I got the same. But still I have not get the 3 kb N-terminal region. Kindly help me friends ! Further I have tried Bio line, Phusion DNA polymerase and Hot start taq polymerase also. Kindly give your advice.
Please have a look at this thread:
https://www.researchgate.net/post/What_are_some_considerations_when_creating_cDNA_from_long_mRNAs
Maybe it can give you some valuable ideas how to address your problem.
Good luck and regards, Christian
6 kb is not long for today's technology standards. Assuming your RNA is OK :
1. Use a good reverse transcriptase (Superscript is by far the best and the only quantitative/reproducible I have tested)
2. Use gene specific primers (one or more antisense primers) instead of random primers, this will already select for your gene of interest at this level.
3. Use a nested primer to the one you used for the RT (+ sense primer) You may need to design more than one pair to get it to work.
4. Try different polymerases. Get some samples or ask in other labs: For example if the Thermo Scientific Phusion High-Fidelity DNA polymerase doesn't work (with both buffers) try Advantage Genomic LA Polymerase Mix . Both are great but work for some genes and not others and Phusion is not very reproducible from one try to the other.
5. Do a gradient to determine the optimal annealing temperature.
6. If you need to clone it (I imagine that is why you want the whole cDNA): use TOPOXL from invitrogen or if everything fails the pJazz from lucigen which has no promoters and you need to be subclone later.
hope this helps
Use SuperScript® III to amplify cDNA up to to 12.3 kb is demonstrated in Figure
https://www.thermofisher.com/it/en/home/life-science/pcr/reverse-transcription/reverse-transcriptase-enzymes/superscript-iii-reverse-transcriptase.html?icid=cvc-superscript-c1t2
Then I should perform a PCR with specific primers and with Thermo Scientific Phusion High-Fidelity DNA polymerase
Try to add DMSO to your PCR mix... it can help you
I agree with Soledad and Marco. Even though a transcript of 5.5 Kb is not that long, your best bet will be the use SuperScript.
Thank you friends thank you very much ! I took your suggestions and I am trying it now. I will be back soon with results. Thank you once again. - Dinesh
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