I am doing PCR using the Hot start KOD polymerase from Merck and l am getting a positive reaction in the NTC repeated from cycle 36-38 in a 45cycle PCR. I am working in a unidirectional manner using separate rooms and Biosafety Cabinets for Master Mix preparation and DNA addition. When l ran the product on a gel it is running further than my 140bp product, appearing as a smear. I am using the lightcycler96 and l am getting a reaction efficiency of 1.95 (perfect amplification is 2). Any comments or suggestions will be hugely appreciated.
Does it only appear in the no-template control?
Most likely it is primer dimer. If target template is not there then primers get bored and start amplifying each other. You will see a different melt curve for this product. Just be sure to check that you don't have this happening in your samples otherwise SYBR green will measure this product along with your gene of interest.
further means smaller ? Than its primer dimers they often occur in NTC at the cycles mentioned. And do not occur when there is template (cause the primers bind more efficiently to the target sequence)
Are you talking about internal probe (TaqMan type ) or SYBR detection? I would not be concern if it is SYBR - anything cycle 36 and up is likely non templated amplification.
It's usual for PCR based on intercalating dyes. Can you see two melting point in positive sample? If yes, add one additional step at your PCR protocol, ( previos to 95ºC DNA denaturation) a fluorescence reading point for 5s or less at 5ºC over the melting point of your primer dimer product. With this approach your fluorescence signal only will be related with your PCR amplicon.
Thank you everyone for the answers, l really appreciate it. Yes Amanda, and it has a unique melting curve profile with a very low signal. Yes Norbert that might explain it. Peter, l am using LC Green to detect the product. Francesc, the positive sample has only one melting point . I have to ask on your suggestion. My amplicon is the rpoB gene (Tb), has a melting point of 89 degrees, so if a have a fluorescence read out at 94 degrees l wont get the signal as all the product will be ssDNA. My current protocol has a read out at the 60 degrees step.
Ian, the fluorescence read should be added in a temperature greater than primer dimer melting point but lower than amplicon melting point. Negative samples have some melting peak?
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