Odd curve of Gel purified PCR product - (Aug/08/2005 )

Hello, everyone

I use Qiagen gel extracion kit to purifiy PCR band of a Gel and then use Nanodrop to concentration of DNA. The concentration is 20 ng/ul in 30 ul EB and the 260/.280 ratio is 1.89, which seems all right. However the curve looks weird. There is no peak at 260 but a big one at 230. I am using the purified DNA for sequencing and wondering if it's fine and why there is no peak at 260..

Thanks a lot

Forforfor

Hi forforfor,

I have heard from other people of the same problem when using the Qiagen Kit, though never had it happen myself. My colleague doesn't believe the spec results for any Qiagen purified samples anymore and runs them on a gel to estimate the concentration instead. She's never had any problems with sequencing (or any downstream reaction that she's done).
I'd recommend running an aliqout out and guestimating it's concentration.

Nicole

Hi

Typically there isn't a peak at 260 for DNA, there is a rounded peak lower in the spectrum, at around 230. The DNA is measured at 260 as the 230 peak is not specific for DNA, however as this measurement is on the side of the 230 peak it can be quite variable.

Bob

thanks, Nicole and Bob.

in a word, you think reading of DNA by nanodrop could be untrustable. Do you have any idea why ? what chemicals has strong absorption at 230?


forforfor

Hi again,

Umm... I don't know. I'm not real good on the "why" for spec's (I *should* be, but I just can't remember these things). I'll be interested to find out though... then I can file it away and forget it next time too

BUT if you know what chemicals are in your sample you could always find out if any of those has an absorbance at 230nm (no, I don't know how, but surely there's some database or something, or the Mercks Index? I'm not sure ... I'm still scared of chemistry )

Nic

Hello,

sombody says that EDTA in the EB buffer has very strong absorption , which interfer the curve. Seems reasonable....


forforfor

Hi again,

Umm... I don't know. I'm not real good on the "why" for spec's (I *should* be, but I just can't remember these things). I'll be interested to find out though... then I can file it away and forget it next time too 

BUT if you know what chemicals are in your sample you could always find out if any of those has an  absorbance at 230nm (no, I don't know how, but surely there's some database or something, or the Mercks Index? I'm not sure ... I'm still scared of chemistry    )

Nic