i have designed a primer for qpcr reaction. when designed this primer i have already consider the general parameters for a good qpcr primer from several sources.
unfortunately my Bcl-xL primer is always display a brigther band in negative control (N) compared to cDNA loading (58). i'm feel pretty sure its not a dimer because the band in negative control has product size around 200bp. and if it has a contamination why it's brighter/thicker than sample with cDNA (58).
if anyone has any idea whats happening with my primer/reaction please help me :(
IN my opinion you have a general problem regarding cycling conditions and/or contamination, sinceyou see different band of different sizes in different "N"-samples (e.g. first vs last column). I think you should try do design a better primer pair and have to test it once more.
I recommend to check the priming strategy again and to design new primers that should ideally Bridge the hughe intron of Bcl-xl.
I will add a Picture.
If you look at the bottom of the Picture, you can see the so called human chain self-alignment. take care not to place a primer into the Region that is also present on the other chromosomes as genomic DNA sequence.
By taking care of these aspects you should be able to avoid false priming of gDNA and your primers can ideally only detect cDNA, but not spourious contamination by gDNA.
I am affraid your positive negative control is based on a contamination with gDNA that got amplified.
I recommend you to design new PCR primers. Definitely, you have dimerization and/or contamination in your negative samples. Looking at your picture, it seems your gel electrophoresis is running too fast or at high voltage in a short period. Why? DNA size marker is not separating homogeneously.
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