.......while i was using a standard kit for some specific mutation......everything was fine but in the no template control when i added the mastermix and nuclease free water i got no amplifications in the mutation channel and the channel specified for internal control (as mentioned in the kit insert) but when i loaded only the mastermix without making up the volume i got an amplification of around Ct 37 (The kit insert doesnt specify this) in the internal control channel ......the kit insert clearly specifies that for NTC there shouldnt be any kind of amplification in the mutation channel or the internal control channel.........can anybody please help me with the same.....are these primer dimers?This is ARMS PCR based assay and it is specified in the kit insert that this internal amplification reaction amplifies a region of exon 2 of the EGFR gene. The primers and probe have been designed to avoid any known EGFR polymorphisms.........
this is a real time PCR based TaqMan probes assay and the total PCR cycle is 45 ......can anybody please help me
Did this just happen once or is it happening repeatedly? I probably wouldn't worry too much about it as a Ct that high sometimes happens in NTC and could be indicative of primer dimers as you mentioned. If you still have them, you could run your samples that you took out of the qPCR machine on a 4-4.5% DNA gel (don't add loading dye) and check to see what the "product" looks like. If it's primer dimers, they should be really small
Thanks a lot Hanna .....I was also thinking in the same direction.....it’s not happening repeatedly .......it happening randomly.......this is primarily happening if only mastermix is placed in the tubes without any DNA and since its purely a ARMS PCR based assay with PCR clamp involving multiple sets of primers(4 to 5 different targets ) so the chance of primer dimer formation with only mastermix is very high where as in the NTC..... in place of template when we are adding Nuclease Free Water there in no amplification (it’s completely clear) as the mastermix itself is getting diluted to some extent so chance of primer dimer formation is getting reduced at the same time as specified in the kit insert (The internal control amplifies a region of exon 2 of the EGFR gene) it’s also not getting any DNA to amplify with the primer of a region of exon 2 of the EGFR gene......I would be really thankful if you kindly relate your opinion concerning the same at your convenience
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