The quality or amount of dna may be a problem. Check the od260/280, increase the extension time by 15-30 seconds per cycle and run dilutions of the dna in case your dna contains pcr inhibitors so maybe try 1/2, 1/4 and 1/8 th the amount of dna and hope that as the pcr inhibitors are diluted the pcr efficiency increases

Thank you :) I will try it

If your fragment is GC-rich, try to add the Q5 High GC Enhancer or 2-5% DMSO.

Use RT-PCR with SYBR Green to see the PCR amplification plot. Use RT-PCR for creation of melting curve to undrstand purity and honogenity of isolated DNA.

Karen A. Darbinyan in my opinion your answer is misleading, genomic DNA cannot be amplified by reverse transcription polymerase chain reaction (RT-PCR) and melting curve analysis for a 7 kb fragment is not feasible.

7kb using gDNA, thats a little bit hard to achieve. Basically, you need to use high quality intact and pure gDNA.

With Q5( using plasmids DNA as template), I usually go with 30 secs at 98, 30 secs at anealing and 30secs/kb elongation( some times +30’’). I would let the late extension ro go for more than 5 minutes. In addition to different amounts of gDNA as input as mentioned above, i also suggest gradient anealling temperatures.

Hi everyone, I am trying to amplify 6200 nucleotides from cDNA, and I am failing miserably. Any other advice in addition to the things said here?

Thanks and have a nice day!