Real time PCR

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Sara.ar
Sara.ar's picture
Real time PCR

Hi,
Trying a primer pair and not getting a nice and smooth std curve! has tried lowring anealing temp with no luck! Any suggestions?
 
thx
Sara

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Sara,
Could you provide more information about your assay? How did you design your primers (which software did you use)? What are you using as template (DNA or RNA) and what dilutions are you using? When you say you are not getting a smooth curve, what do you mean? Are you running SYBR Green assays, and if so how does your melt curve look like?
 

Sara.ar
Sara.ar's picture
using universal probe library

using universal probe library with cDNA from parts of brain. Dilutions starting from 1ng and diluted 10x up to 5 dilutions...My curves look like almost an straight line instead of a nice amplification curve which platue eventually! No SYBr green.

Ivan Delgado
Ivan Delgado's picture
 

 
Unfortunately I do not have experience with universal probe libraries. My first guess would be that somehow the assay is not working, but since you do not have access to the sequence of your primers (and probe) it is hard to troubleshoot. I also assume your cDNA is commercial so that should not be a problem. If you made the cDNA, then it would be a good idea to determine that it is good cDNA and it is not degraded. 
My suggestion would be to contact Roche (or the maker of the probe library) and ask them if they know the source of this type of problem. Alternatively use an assay you know works to make sure the cDNA is of good quality.
Good luck
 

Sara.ar
Sara.ar's picture
Thanks Ivan

Thanks Ivan

Touchstone42
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http://www.caister.com

http://www.caister.com/molecular-biology-blog/2008/10/pcr-troubleshooting-primer.html
The recommended primer concentration for PCR is between 0.1μM and 1μM of each primer. The use of higher concentrations of primers can have the following effects: If the primers are capable of forming dimers, raising their concentration only results in the creation of primer-dimers and does not improve the amplification of the desired PCR product. Primer-derived oligomers will possibly contaminate the reaction. If the primers do not form primer-dimers, it is likely that raising the primer concentration will lead to non-specific primer binding and the creation of spurious, undesirable PCR products. Raising the primer concentration does not therefore cause an increase in the effective concentration of the primers. Low primer concentration generally ensures cleaner product and lower background. However, to amplify short PCR target sequences, careful calculation of the optimum primer concentration is required. For example, if the target fragment length is 100bp, a greater number of PCR product molecules is required to provide a specified amount of amplified DNA (in nanograms) than for a larger target fragment. In order to generate the required number of PCR product molecules, a greater number of primers may be needed. Therefore, concentration of primers higher than 1μM may be necessary, and desirable, for short target sequences.
http://www.caister.com/molecular-biology-blog/2008/10/pcr-troubleshooting-primer.html