It could be non specific amplification or contamination. If it is contamination it comes from template DNA, because in a 5 probe you got correct size. First at all check it, also I will recommend you to increase the melting temperature. If not works you should design new primer set. Good luck !!! 

Hi Chaitanya,

The first band of alpha 5 is from same cDNA sample??

Hi,

There is genomic DNA contamination in your cDNA. The Gabra1 primers are on exon 9-10 and there is a 1617pb long intron in between. By designing new primers you may easily overcome this problem.

For Gabra5 you have other problems. The forward primer sequence only partially fit to the target (TGGAAGCAGC) and the reverse primer does not fit at all.

@Pankaj Yes, it is Mr. Pankaj!

Hi,

I think that your big band is due to genomic DNA contamination. To avoid getting this band you should treat your extracted RNA with DNase to remove genomic contaminations (there are several ways of doing this, check commercial kits and ask if you need help)

Regarding the problem of alpha5 suggested by Mr Ayata you can sequence the PCR product to check if that is your desired product.

Also from your picture looks like you could improve PCR conditions by trying a gradient PCR.

Good luck!

@Alfonso Saera-Vila thanks a lot.....much appreciated

Hi Chaitu. This is the gel you submitted for your MSc project in my lab over two years ago?

At the time we discussed (and others have correctly pointed out) that there was gDNA contamination in your reaction, which likely accounted for the large amplicon in your alpha 1 PCR. The alpha 5 primers are 100% specific to the target transcript (accession NM_017295), a simple BLAST search will show you that - remember the reverse primer is the reverse complement sequence (although orientation will not matter in a BLAST analysis). If you remember correctly, the alpha 5 subunit gene is not well expressed in that region of the brain, so weak amplification was expected - plus this PCR was only completed by yourself once or twice with no optimisation - due to time constraints in your project.

If you still want to talk about this data, I would appreciate it if you would contact me directly.

Thanks