2 replies to this topic

[Serhiy Pylawka]

I am running regular PCR with primers I designed for my RT-PCR spanning over an intron from different exons.  The results are very confusing. I do get my expected  gemonic bands (intron + exon); however, I also get band I would get from cDNA (exon, minus intron).  My genomic bands presence is inconsistent but my cDNA band extremely consistent.

I tried using 5'UTR primers, primers spanning over many introns with no avail. Any suggestions please, please.  

Serhiy

[chrisbelle]

Serhiy Pylawka, on Jan 28 2010, 01:48 AM, said:

I am running regular PCR with primers I designed for my RT-PCR spanning over an intron from different exons.  The results are very confusing. I do get my expected  gemonic bands (intron + exon); however, I also get band I would get from cDNA (exon, minus intron).  My genomic bands presence is inconsistent but my cDNA band extremely consistent.

I tried using 5'UTR primers, primers spanning over many introns with no avail. Any suggestions please, please.  

Serhiy

What exactly do you want to amplify? The intron only or over the exons including the intron? Since you are doing RT-PCR (I assume reverse-transcription) you are not supposed to get the genomic DNA bands.

1. You have DNA contamination in your RNA
2. If your band from cDNA is your expected then your primers are fine. Deal with the DNA contamination.

Chris

[stylothecancer]

chrisbelle, on Feb 3 2010, 02:25 PM, said:

Serhiy Pylawka, on Jan 28 2010, 01:48 AM, said:

I am running regular PCR with primers I designed for my RT-PCR spanning over an intron from different exons.  The results are very confusing. I do get my expected  gemonic bands (intron + exon); however, I also get band I would get from cDNA (exon, minus intron).  My genomic bands presence is inconsistent but my cDNA band extremely consistent.

I tried using 5'UTR primers, primers spanning over many introns with no avail. Any suggestions please, please.  

Serhiy

What exactly do you want to amplify? The intron only or over the exons including the intron? Since you are doing RT-PCR (I assume reverse-transcription) you are not supposed to get the genomic DNA bands.

1. You have DNA contamination in your RNA
2. If your band from cDNA is your expected then your primers are fine. Deal with the DNA contamination.

Chris

Hi Serhiy Pylawka,

Agree with Chris.

Actually I am also not very clear with what you wish to do when I read you post.