I designed a primer with high Tm temperature (74, 70)
[I could not find a better primer. In addition, before PCR, I have to do RT-PCR to convert RNA to c DNA]
1. What kind of PCR do you suggest me? Is TD-PCR proper?
2. If yes what temperature would you recommend for star of gradual decreasing of temperature?
Thank you
Hi Marjan,
I dont´t undarstand your problem but i think RT-PCR is the better technique for you.The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression.
What is the name of your primer?
See this site:
http://www.dbbm.fiocruz.br/helpdesk/mbiology/PCRcurso.pdf
General Tips for TD-PCR:
1. Keeping all reactions cold until thermal cycling starts is crucial to avoiding non-specific priming even with TD-PCR.
2. A hot start setup is preferred. Since the main aim of TD-PCR is to eliminate non-specific interactions during the initial cycles, it is important to use a hot-start set up.
3. TD-PCR can address problems with monoplex reactions better than multiplex reactions.
4. Total number of PCR cycles, including the touchdown phase should be kept low (below 35). Too many cycles will lead to appearance of non-specific bands in the gel.
5. An extra 1 min denaturation cycle at 96C or 97C may be extremely useful for difficult templates.
I hope I have helped you
João Pais
Jaop Pais
I designed a primer with high Tm temperature (74, 70)
[I could not find a better primer. In addition, before PCR, I have to do RT-PCR to convert RNA to c DNA]
1. What kind of PCR do you suggest me? Is TD-PCR proper?
2. If yes what temperature would you recommend for star of gradual decreasing of temperature?
Thank you
Well if you go for TD-PCR your initial annealing temperature will be 80-84 which I dont think will be good idea
However what you can do here is use only 2 step method ... melting and then annealing and extension combined in one step ... this may work for such cases
Hi all,
I'm dealing with the primer which is 84 °C for forward primers and 65 °C for reverse primer. Is it possible to succeed PCR?
Reina
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