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PCR and UV spectrophotometry

quantitation PCR UV

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3 replies to this topic

#1 Thaqifah Salihah

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Posted 15 February 2012 - 09:02 AM

hi,

I'm fairly new at working with DNA. so, any help is greatly appreciated.

i'm trying to quantify fairly degraded DNA. and I'm going to use the electophoresis method (after running my sample through the PCR process). I'm also going to quantify the sample by using UV spectrophotometry. I'm not sure whether I could use the PCR-ed samples and quantify them as well using UV spectrophotometry. because someone told me that you can't use PCR products for UV spectrophotometry, you have to use your original samples. why though? because I've read that UV spectrophotometry won't work with very small sample, and I want to amplify them first before quantifying them....I'm clearly out of my depth here. so help!!!!

#2 mdfenko

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Posted 15 February 2012 - 09:28 AM

you have to purify the pcr product before spectrophotometry because if you don't then you'll read the dntps as well as the dna.

Edited by mdfenko, 15 February 2012 - 09:29 AM.

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#3 leelee

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Posted 15 February 2012 - 05:36 PM

It is a little unclear to me exactly what you are trying to do.

If you want to quantify the amount of DNA in your original sample, using PCR will be useless. The PCR will amplify your DNA many many times over, and this won't tell you anything about your original sample.

If you just need to quantify your PCR product, then yep as mdfenko says you'll have to purify if first before using the spectro

#4 ickypimp

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Posted 16 February 2012 - 03:36 PM

If you want a more accurate quantification then using an intercalating fluor like pico green will give you a better result, if using UV spectroscopy then only rely on this for a "quick and dirty" method of quantification and only use it for comparing products of similar sizes (read up on the hyperchromic effect and base stacking)





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