Hi Nikita Sharma

You can check and follow a few things.

First check the primers have any other non-specific homology with Drosophila genome. If there is, you may need to change the primers. Also, check if the cDNA has the complementary primer sequence. If everything is fine (5'-UTR, 3'-UTR, introns), go further.

Q5-HF is an excellent choice but sometimes it needs to be standardized according to different PCR reactions. You can start with a gradient temperature PCR (different annealing temperatures, for instance, from 60C to 70C).

For long amplification, you also need to increase the extension time, i.e. 40s-50s/kb. I used 2 minutes and 30 seconds extension time for a 4 kb amplification.

If the primers are long, have complex or secondary structure, or higher Tm, 1.1M Betaine significantly reduces the annealing temperature and enhances amplification.

All the best!

Thank you for your response. I rechecked that the primers have no homology to any other region in Drosophila. I tried gradient PCR as well but it’s not working in this case. I’ll try once with Betaine and see if it works for me. Thanks a lot!

Sometimes running a smaller volume (20-25 ul) will help improve amplification of larger products. The reaction heats/cools more efficiently with a smaller volume and that can be helpful.

Double check that you can amplify something else (something smaller) from your gDNA as a way to make sure all the reagents and the gDNA are good.

Amy Klocko Thank you for your response but I used 10ul reaction and I am using cDNA for amplification and not gDNA. I amplified another smaller gene from this cDNA which was perfectly fine.

My apologies for not noticing that you are using cDNA. It’s a large size transcript so you may need to optimize the cDNA synthesis parameters (longer synthesis time).

Or, try for a tissue or time points predicted to have enriched expression of your gene.

do your primers work in gDNA? Any possible chance of alternative splicing?