Hi, There is no difference. But it was suggested to use primers by product size of 200-400 for real time.
Best
Dear Mohd,
Regarding your question, you must determine if you mean that the same primers can be used both for qPCR and endpoint PCR, or if you mean that you design the primers with the same way. The primers that work on qPCR, can actually work on endpoind PCR. We test the primers for qPCR by performing gradient-endpoint PCR before. Just keep in mind that you must use the same type of template (cDNA or DNA) in each case.
However, the rules for designing primers for qPCR are more restrictive. It depends on which detection method you are going to use (Taqman probes of SYBR Green Dye). With SYBR green you must be absolutely sure about your primers. The lenght is usually smaller (80-150bp) and the Tm is higher than the endpoint PCr primers (Usually 55-60).
You must clarify your question and your experiment so tou give you more answers.
Good Luck
There is no difference. For a reliable quantification by real-time PCR it is very important that the primers really amplify exclusively the specific product and that this amplification runs with almost optimum efficiency. These criteria are often not so important in conventional PCRs, where the main target is simply to get some amplified specific product at all (rather than a precise quantification of initial concentrations of the target sequence).
Agree with all of the above. In general for intercalating dyes (like SYBR green) you really want to use smaller amplicon size as mentioned above (80-150bp), plus you have to be more careful about efficiency & right GC/TA mix, since everything shows. At this point, I am just beating a dead horse, though.
Hi Mohd!
Although there is no difference between Real Time PCR primers and normal PCR primers, I recommend you to first optimize your primer design and perform a gradient PCR. It is important that the primers amplify with precision the product size.
Good luck!
It is very important that you have good primer design for real-time PCR. There are many online guides which are quite useful. One of the main issues is to avoid primer dimers as these will interfere with quantification. Again there are online tools to check for both primer pair and self dimers (e.g. http://www.operon.com/tools/oligo-analysis-tool.aspx). When I am designing primers I design 2 or 3 sets and test them to see which are the most efficient. As others have said, 80-120bp is optimum, 150bp maximum I would say.
I have very good experience with the free online program by MIT. I design for 100-200 bp products.
http://bioinfo.ut.ee/primer3-0.4.0/
The primerblast program from NCBI uses the same interface but also blasts the sequences at the same time. On Biorad real time PCR machines I usually have great efficiency at the first go. For cDNA I always design intron spanning primers.
hi, Dr. Mohd
i agree with most above , there is no different. only different between the two process is in the detection chemistry which found only in real time pcr
because real time pcr is in its step is pcr put detected through the progress pf the reaction not at the end of it like normal pcr
there is no different. only different between the two process is in the detection chemistry which found only in real time pcr because real time pcr is in its step is pcr put detected through the progress pf the reaction not at the end of it like normal pcr
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