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Issue with standard curve for qPCR for CHiP


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#1 Maxwell

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Posted 04 November 2010 - 08:00 AM

Hi,
I'm new to this forum, so if I haven't given enough info about my problem just let me know. I'm attempting to make a standard curve with a set of primers I'm using to analyze CHiP samples with. I'm using a sybr green mastermix also. My issue is that I need my standard curve to extend from Ct values of about 20 to 35, and I know from previous data that my CHiP samples will most likely fall in that range. I've used serial dilutions of a CHiP input sample from one of the cell lines I'm using in the experiment, and my range of Ct values extends from 26 to 36. I've doubled the amount of template in each reaction (1uL more template, 1uL less ddH2O) with the new range of Ct values being 25 to 35. I am aware that primer dimers can form with sybr green, but my thermal conditions for the reaction will melt the primer dimers (and not my product) before the samples are read. It has been explained to me that I cannot make the starting concentration of my Jurkat input more concentrated, so that is not an option. Thus I am left with CHiP sample Ct values that fall outside of my standard curve, and therefore can't be validated without extrapolation. I would very much appreciate any advice or pointers on what to do or possible next steps to take.
Best,
Maxwell

#2 tea-test

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Posted 04 November 2010 - 09:47 AM

Hi, you could try to use diluted pcr products from a previous pcr reaction as starting template for the standard curve. you should column purify the pcr product and be careful not to contaminate your samples.
tea-test: The artist formerly known as Ned Land




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