Hello there!
Does anyone have experience with absolute quantification using SYBR green kit for bacterial dna ? And plasmid as positive control, Could tell me the steps and his advice that i have to do to make the standard curve?
thanks in advance
The paper recommended by Wilhelm is the best reference to prepare standards for absolute quantification. However, my suggestion is to avoid SYBR green method and use probe-based chemistry for more accurate results.
best wishes.
Probe-based methods are more expensive and more error-prone (additional oligonucleotide that can interfere with primers, unspecific amplification products are not detected and require post-PCR processing) - but they are not more accurate. Given that one amplicon molecule binds 10-20 SYBR Green molecules, each of which fluoresces, while only a single fluorescent molecule adds per signal per amplicon molecule in a probe-based system, SYBR Green should also have a better signal/noise ratio (depending a bit on background and fluorescence efficiency) and might therefor give a higher accuracy.
Probe-based detection systems are advanatgous to detect (not to quantify!) sequences in very low concentrations (where the amplification of unspecific producs can mask the SYBR Green signal of the correct product), to discriminate sequence variants (SNPs), and to multiplex. These advantagous are irrelevant in standard singleplex qPCR.
Jochen Wilhelm, Thanks a lot for the information. Could you please share some more literature on the advantages of SYBR based methods. In fact, they are relatively less expensive, easier to optimize and S/N ratio is better. However, in case of virus detection and quantification, researchers prefer probe-based methods due to low template conditions, as mentioned by you. I'd like to get more info on this aspect.
regards,
I don't have any literature at hand, I would have to look for it.I am sure you will find it yourself after a look into PubMed or even Google.
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