Hi

if you want to do qPCR in regards to the DeltaDeltaT analysis, due to performance of most taqs, dur to varying qualities in samples, due to amplification errors and why not presence or absence of variants in your targeted sequences or in the primers...the best amplicons should be around 100 pB. if qualities of your samples are varying, some of them will be amplified and other not, giving you false conclusions in your analysis. In addition, if you do qRT-PCR on RNA or cDNA, taking overlapping primers (almost for one) is a key point in your protocol for designing primers.

fred

The normal maximum is 200 bp.  Too large and the amplification is not efficient.  You could always try it, but you will need to be VERY thorough with your quality control and testing if you want to publish results.  You really need to design primers for a smaller product.

Dear Virlana,

You will certainly be able to amplify a 670bp fragment in qPCR, but you will likely obtain inaccurate results. It is not altogether clear why you would want to amplify such a large fragment for qPCR. I recommend that you redesign your primers to amplify a fragment of about 100 bp.

Best.

Thank you everyone for your answers! I am not doing RT or anything that requires absolute or relative quantification - I basically just need my target amplicon to send for sequencing. I was asking about the qPCR route rather than the PCR route because in our lab in the past, qPCR has worked better for low template concentrations.