incorrect/smaller band size for real time PCR - (Nov/06/2010 )
Are real time PCR products shorter in size as compared to standard PCR ? I am expecting a band size of 248 bp but I am getting 100bp on a gel. My NTC is negative and there are no primer dimers. What is the cause for getting a smaller band size ? Any advice would be appreciated.
Real time PCR products are not smaller than regular PCR products. Real time PCR assays are typically designed as smaller amplicons, but if you designed it to be a 248 bp amplicon, then it should run around that size on a gel.
Of course it all depends on what you are comparing it against. If you are running a size ladder that uses a different loading buffer than the one you use to load your real time PCR product, and you did not clean your real time PCR product (i.e. your real time PCR reaction still contains SYBR Green), then your PCR product will likely run much different in size on a gel.