very bright non-specific band - (Jun/01/2008 )
Hi,
Im hoping to amplify CD-46 gene with F and R primers of 53bps each. There is a very bright band (180bp)but its not of the expected size(260bp). There is no other band on the gel. I've re-re-checked the primer sequence with the literature. Will increase in the annealing temperature make any difference?
Im hoping to amplify CD-46 gene with F and R primers of 53bps each. There is a very bright band (180bp)but its not of the expected size(260bp). There is no other band on the gel. I've re-re-checked the primer sequence with the literature. Will increase in the annealing temperature make any difference?
Have you blasted your primers to make sure they are specific to cd46? You can use the UCSC website to double check your product size.
The band you see might just be primer dimer 
You can try to do a step PCR increasing the annealing temperature over the cycles just to see if you get better results Start with a low annealing temperature to allow even weak annealing to occur and the^n each 5 cycles you increase the temperature by 2°C
Do you try to amplifiy from Genomic DNA ? How much DNA did you put into the reaction ?
In my experience, i would redesign the primers with different 3` ends (shorter or better longer than before). If you're getting a non-specific product this strong, it is difficult to get the primers to not bind to it.
You can try to do a step PCR increasing the annealing temperature over the cycles just to see if you get better results Start with a low annealing temperature to allow even weak annealing to occur and the^n each 5 cycles you increase the temperature by 2°CDo you try to amplifiy from Genomic DNA ? How much DNA did you put into the reaction ?
Im amplifying from genomic DNA by taking about 400-500ng per 50 microliter reaction mix. they cant be primer dimers ...roughly 200bps of primer dimer??
I have blasted the primers now. and the website says that i should get products of a size of 270bps!
Why are your primers so long? Does the gene have very low GC ratio? What temperature conditions do you use?
New 3` ends on both primers is still my #1 suggestion but another suggestion is to digest the genomic DNA with a battery of restriction enzymes not present in your target. This may digest away the secondary template and prevent it from becoming amplified, hence favouring yours. Or rather than a battery of restriction enzymes, choose a restriction enzyme with a simple common recognition sequence not present in your target - this will digest away more potential secondary targets.
It would also be good to sequence the wrong fragment just to see what it is. This may sound like a waste of time (technically yes) but then you can identify exactly what it is and why the primers are amplifying it (i.e. what sequence they are annealing to). It may be that one of the primers is amplifying both ends of this false target and a simple change to one primer will prevent it. Maybe your primers are fine but you're amplifying a deletion of your fragment (unlikely but you never know). It's good to know exactly what is going on and the sequence of the fragment will provide invaluable clues.