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standards for absolute quantification qPCR

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#1 starvingstudent



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Posted 19 April 2010 - 06:21 PM

Hello all,
I am doing absolute quantification and have generated standards which are basically my gene of interest inserted into a plasmid. I linearized my plasmids with a restriction enzyme. I then determined the concentrations of the linear plasmid and make 8 10-fold serial dilutions. For the past few trials, the Cp values of my standards seem to plateau after the 6th dilution (for example, here are some results for my standards from a recent run)

Pos Cp
A1 12.48
A2 15.27
A3 17.51
A4 18.16
A5 18.33
A6 21.08
A7 17.67
A8 18.15

This is a problem because the standards don't seem to be covering the range that I need and my samples fall beyond the range. Has anyone encountered this problem? I thought it might have been the plasmid, but I re-grew new plasmids and that didn't seem to help. Could it be the primers?

I would appreciate any tips/suggestions you may have! Thanks in advance!

#2 phage434



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Posted 20 April 2010 - 10:53 AM

I strongly suggest doing a no-template control. I would guess that it also would amplify, meaning that you have non-specific amplification, or worse, contamination of one of your reagents with small amounts of template. You could also be doing the 10x dilutions incorrectly. Be sure to use new tips each time, and be very careful of accidental carryover of high concentration material to low concentration tubes on pipet barrels, etc. Use barrier tips if possible.

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