Hi Poshan,

cDNAs are more difficult to PCR-amplify than plasmids DNA. Here are my suggestions to sort out this issue:

* do a nested PCR and if that still does not work, then you can

* split your fragments (1.7kb and 1.3kb) in two smaller fragments (less than 800bp each) and ligate them afterwards (dont forget to add overhangs while designing your primers in this case)

Best of luck

Steve

Try to performed the PCR conditions

Dear Pochan,

As you have noted in your question, the RT protocol is working fine. I am suspecting the challenge might be at cDNA step particularly the expression level of your gene of interest. Depending on the position of your PCR fragment (either near the 3' end or 5' end of the gene), the expression of your PCR fragment differs depending on the primer you are using during the reverse transcription in your cDNA synthesis step.

Here are my suggestions:

1. Use gene-specific primer in your reverse transcription step

2. If that doesn’t work, use random hexamers or random hexamers plus oligo dt in making you cDNA

Hope it works for you.

Regards,

Israel

Momina Jamil: Yes that could work if everything else fails.

Momina Jamil

1: Verify that your gene of interest is being expressed using transcriptomic data sets ( if publicly available)

2: If you are sure that your genes express, perform a gradient PCR on the cDNA.

Best.