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Concentration of cDNA from unknown quantity of RNA by mistake


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#1 naa

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Posted 19 September 2013 - 07:21 AM

Hello,

I would appreciate it if someone can help. 

 

Is there any possibilty to find out what is the concentration of cDNA transformed from an unknown quantity of RNA (1ug or 2ug, protocol paper lost) knowing that the quantity of cDNA is limited (few uL) and no more RNA is available.

Thx 



#2 jerryshelly1

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Posted 19 September 2013 - 08:43 AM

You don't have any information? You should be able to put it on a nanodrop and read the concentration. Just place a small amount 1uL on the nanodrop, read the sample and then take the liquid back.



#3 naa

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Posted 19 September 2013 - 11:31 AM

I did got a value of 165.5 ng/ul so what does this mean a real concentration of 100ng/ul? or 50ng/ul



#4 jerryshelly1

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Posted 19 September 2013 - 12:57 PM

It is hard to say without using a DNA specific dye. You would be detecting both DNA and the degraded RNA.  What is your 260/280 ratio? Can you spare 1uL and run your sample on a fluorometer? If we are just ballparking it I would just subtract 50ng and say you have an estimated concentration of 100-115ng/uL.



#5 naa

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Posted 19 September 2013 - 01:23 PM

the ratio 260/280 is 1.53 but didnt understand what can we use it for. 

thk u for ur help. i appreciate it



#6 naa

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Posted 19 September 2013 - 01:27 PM

i know its for the contamination.. :/



#7 jerryshelly1

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Posted 19 September 2013 - 02:48 PM

Yeah. Not a very informative method, but it can tell you the purity of the DNA/RNA. Absorbances of 1.7-1.9 usually signify purer DNA. You can sometimes use this as a close estimate to the relative amount of DNA, as compared to RNA; however, it is all relative. You can't really base it on this number, especially since cDNA protocols contain a lot of contaminants.

 

If you can spare some of your sample, run it on a fluorometer. Fluorometers can usually detect small ng amounts of DNA, so you could possibly dilute a small amount (<0.25uL) of your sample.

 

Sorry I don't know have additional recomendations



#8 naa

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Posted 19 September 2013 - 10:26 PM

Thx anyway 






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