real time pcr problem - (Aug/31/2005 )
hi, i have a question about real time PCR. in my sample and my NTC reactions i see peak. they have got the same Tm however when i run it in agarose gel i see the an expected size band in the sample reaction and not in the NTC. how can i solve this problem??? can it be a result of low concentration of cDNA???
What cycle is your peak coming up? Also does it look sigmoidal? Are you using taqman or sybr green?
i checked the pcr products again in the gel and it seems that both reaction gave the same product (my mistake). however is there any other explantion than dna contaminaion???
i use sybergreen, the reaction looks sigmoidal and the peak is in cycle 35.
Could be primer dimers or it could be contamination. Really the only explanations, you need to do the controls to figure this one out.
For your NTC, you should see one peak which represent primer dimer. The peak will be located at the lower temperature compared to your positive control.
So any sample which do not amplified or not amplify in exponential way, you will expect to see a peak like primer dimer.
Since your NTC peak is same as your positive control, and you seen a band by runing gel, that is a typical DNA template contamination problem. The DNA contanmination is either occur in your primer solution, H20 or in your reagent and you pipette as well.
i suggest you to autoclave your pipette, use filtere tips, use new aliquot of primer, and new aliquot DNase free water. if the problem persist, change your reagent.