Will increasing my DNA template increase the efficiency of the PCR reaction? I have currently got 10 ng of template in my reaction mix. My boss has asked me to try adding 50 ng of template to increase the reaction efficiency? Any feedback from the science community?
Amount of template is one of the factors that can influence efficiency of your PCR reaction. My answer is: not necessarily-more does not always mean better. Remember that your DNA/RNA preparation always contains some protein and salt contaminations that may inhibit polymerase activity. Its robustness is also restricted. Check precisely cycling conditions adequate for your enzyme, cycler(!) and primers Tm.
And finally plot the efficiency curves by running serial dilutions of your samples at different temp. Even if you don't know the equation your qPCR software will do it for you.
Hope it will help!
Amount of template is one of the factors that can influence efficiency of your PCR reaction. My answer is: not necessarily-more does not always mean better. Remember that your DNA/RNA preparation always contains some protein and salt contaminations that may inhibit polymerase activity. Its robustness is also restricted. Check precisely cycling conditions adequate for your enzyme, cycler(!) and primers Tm.
And finally plot the efficiency curves by running serial dilutions of your samples at different temp. Even if you don't know the equation your qPCR software will do it for you.
Hope it will help!
Hi Lisa,
As explained above by Magdalena, solely increasing amount of template may not necessarily affect the efficiency of amplification. it depends upon several factors...Assuming the other components (inhibiting the reaction) are at minimum..it would primarily affected by primer template interaction. Try to increase the optimum conditions for the assay first.....Increasing only template may give you some more product but may also result into some non-specific amplifications..
bye
Increasing template concentration will be based on amplification efficiecy
1. template quality: poor quality of template inhibits the PCR amplification
2. Amount of primer getting used in PCR: some cases primer is not getting used efficiently in such cases either decreasing primer or increasing template concentration will help to carry out qPCR
3. If above two are not the limiting conditions then try to work out amplification efficiency.
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