I am performing a qPCR standardization of some MGB probes. I have tried just a few times but I don't want to waste reagents, every time I tried the assay, there is a jagging line.
I am thinking that could be that the Reaction mix is not enough for the amplification or maybe I am doing wrongly the setup of the machine.
Do the MGB probes need an especial or different reaction mix?
These are my conditions:
Real-time PCR System: One step Plus - Applied Biosystems
Probes:
Probe1: [FAM] GGG TTT AAA GGG [MGB]
Probe2: [CY5] GTC AAA TCA TCA TGC C [MGB]
Primers:
1F: GTCAGCTCGTGTCGTGA 1R: CCATTGTAGCACGTGTGT 2F: AGCAGCCGCGGTAAT 2R: CTAAGCATTTCACCGCTA
Reaction mix:
Taqman universal master mix (applied biosystems): 12.5 ul
Primers 10uM: 0.5 ul
Probe 10 uM: 0.31 ul
NFW: 6.69ul
Sample DNA (5ng/ul): 5 ul
PCR conditions (As reference article indicated):
Holding stage:
50°C (2 min)
95°C (15 min)
Cycling stage:
95°C (30s)
60°C (1 min)
Did you perform the experiments for optimization of probe concentration? FAM probe has three G in 5' end (guanine in 5' end of a probe should be avoided). Your pictures show curves for FAM, Cy5 or both?
Hi Jorge,
the first thing you should do is to check for amplification specificity in testing your primers couples for in vitro PCR tools as in UCSC (http://genome.ucsc.edu/cgi-bin/hgPcr) or NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) with the rights species and genomes.
Once this has been done, eliminate all causing points one by one.
fred
Hi Jorge,
I haven't performed qPCR with Taqman probes and only with SYBR green. However, I have seen such jagged lines during my runs only when my template DNA has some contamination. In most cases, I was able to rectify this issue by performing a phenol:chloroform extraction or simply ethanol precipitating my template DNA. I don't know what your template is or how you prepare it but it could be worth a try ensuring there are no contaminants (could be organic solvents or salts) present in it. Hope this helps!
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