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ssDNA Quantification


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#1 alan6017518

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Posted 28 May 2009 - 07:38 AM

Hi all,

I am a new user to this forum, this site actually makes me feel like I am not alone^^ Anyways hope I can get some help with my new job. I am a rookie DNA-RA. I am working on DNA probes to capture the CpG island for sequencing.

I have been learning how to make probes for the past month, the last step of the protocol is to quantify the concentration of the probe sets by running a TBE-Urea gel with 90-Mer with known concentration, and taking the image and based on the intensity of the band we estimate the concentration.

However I don't find this method to be accurate, and I want to see if you guys have any suggestions about quantifying the concentration of the ss-DNA. Our lab have a nanodrop machine, but other probe-makers do not use it to quantify (I don't know why) and I searched online and see a product from Invitrogen called the Oligreen (Searched here but got no results)

So what do you guys think? Any help would be appreciated

Thanks
Alan

#2 mastermi

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Posted 28 May 2009 - 10:21 AM

The nanodrop has a algorithm for ssDNA. If you know the size of your fragment, you should probably be able to calculate your mol, although I am just doing this for RNA...

#3 alan6017518

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Posted 28 May 2009 - 12:36 PM

Do you think the nanodrop is the most accurate among the 2 other methods (Oligreen, Image based analysis) ?

#4 Sonia

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Posted 04 June 2009 - 05:30 AM

I'm also using nanodrop for cDNA quantification and have a question. The RT-NTC conc. is around 600ng/ul results, while the sample conc. is 1200-1300ng. Does it mean the real conc. of cDNA is around (1200-600=600ng/ul)??

#5 mastermi

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Posted 07 June 2009 - 12:54 PM

Do you think the nanodrop is the most accurate among the 2 other methods (Oligreen, Image based analysis) ?


I don't know Oligreen, but I think that Nanodrop would be more accurate than gel-based quantification.


Sonia: I'm also using nanodrop for cDNA quantification and have a question. The RT-NTC conc. is around 600ng/ul results, while the sample conc. is 1200-1300ng. Does it mean the real conc. of cDNA is around (1200-600=600ng/ul)??

What means NTC?




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