Syber green - length (Feb/08/2008 )

hi all,

Need to clarify some doubts, I have a primer and probe set available to me, this real time can be done using a taqman probe..


I do not want to spend that much money and wish to use the same primers for a syber green pcr, can that be done? So essentially I would be using the primers from the taqman, and not the probes? is that possible.... If so what are the things I need to do before i can get to the actual assay, I have heard that you need to standardise the melting curve for syber green??

Also the size that I am amplifying is around 100bp, can syber green work in that small a amplification????

can someone help me in this matter

thekid

100bp PCR product ishould be ok for SYBR. Check, if genomic dna is detected with that assay if you do not use a probe. Taqman assays are often designed with the probe on the exon/exon boundary. so if there is a short intron between you would detect it with sybr. concerning the melting curve: make sure, there is only one single peak in your samples visible and furthermore check on a gel for a single band.