ssDNA Quantification - (May/28/2009 )
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
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...
Do you think the nanodrop is the most accurate among the 2 other methods (Oligreen, Image based analysis) ?
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)??
alan6017518 on May 28 2009, 10:36 PM said:
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?