PCR amplified product size - (Mar/26/2013 )
i Have a question about pcr product size...My supervisor said that I need to confirm my pcr products either they are target amplification or PCR artifacts. He asked me to check the calculated amplified size to see if the PCR size is correct....Im not quite understand bout it...How to check the calculated amplified size to see if the PCR size is correct??
Go to pubmed and find your gene. Go to clustalw or any equivalent sequence aligning site. This program will show you where your forward and reverse primers will bind to your gene of interest. Obtain nucleotide expected size.
Or, just transfer your gene to microsoft word and search for your primers.
but im only using foward primer and oligo dt primer and not using reverse primer since im working with rt-pcr (3' race)...so,how to know where the oligo dt primer will bind to the sequence of the gene?
As the name implies, Oligo dT's will bind to its complement, adenine. Polyadenylation is found on the 3' end of a mRNA. The DNA that results from your oligo dT synthesis will be a complement (cDNA) of your mRNA. If you are only using a forward primer, then find the size from your forward primer to the end of your gene segment.
I just reread your post after typing. Are you using both a forward primer and a oligo dT for plain PCR, or are you using an RNA specific primer plus your oligo dTs. If you are using an RNA primer and an oligo dT primer with your extracted RNA as a template for RT-PCR, this is a definite no-no.
Please be more specific.
im using cdna as template for rt-pcr....im using both gene specific primer and oligodt primer....
If you are using cDNA, oligodT would not bind, unless you have a very rich adenine region within your gene of interest. I would get on the manufacturers website (whoever you got the oligo dT from) and try to determine what the length is for your oligo dT primer. In my opinion, the design of this experiment is not well thought out. You should not be using an oligo dT primer to do determine if your gene is present. Adenine/Thymine only base pairing can be fairly non-specific and give you random products depending on the length of your oligo dT primer. Spend the extra $5 and get a reverse primer specific for your gene.
No matter what you do, you will more than likely always get different banding patterns in your PCR products (if using the method you described).
If you need help designing a new primer, post the gene and the Tm of your forward primer. I would be happy to walk you through designing a reverse primer.