A260/A280 at 1.3 - HELP (Nov/05/2009 )

Hi Maddie, what is your DNA dissolved in when it is measured on the spec? A slightly alkaline buffer solution can make the values more accurate; see this paper: BioTechniques 22:474-481 (March 1997)

Also, sometimes my extracts have particulates which can lower the 260/280 values; if you measured A320 and it is high (should be around 0.00), that may be the cause. I've removed them by centrifuging the DNA for a few minutes and transferring the "supernatant" to a new tube

mdfenko: I'm concerned I might have too much proteins left (probably mainly collagen and/or degraded collagen) and I was wondering if I should make another round of proK digestion.
I'll probably try. I know that something in my extracts inhibits PCR and since I removed the humic acids, I now suspect collagen.

Chason: I decalcify the bone powder with EDTA 0.5M, a bit of detergent and proK. Then I concentrate and clean up with the MinElute kit from Qiagen. I don't have any particulate (that I can see). I thought I read that EDTA could give trouble for the A230 but not the A260 or A280. Is that right?

About the particulates, they may not make your DNA cloudy, but there may be enough to interfere with your absorbance readings. You can find out if there are particulates by taking a reading at A320 (not 230); if it is above 0.01, I'd centrifuge the DNA and read the supernatant absorbance

The solution you dilute the DNA in can also affect the readings. If your DNA is in water when you read it on the spec, the 260/280 ratio will tend to be lower than if it is in a slightly alkaline buffer

These may not be your problem, but they are things to check before doing more digests.

Edit: I don't know if EDTA affects A230 readings

you can try a phenol:chloroform:iaa extraction and ethanol:sodium acetate precipitation to remove any proteins that may remain in your dna solution.

you can determine if edta will effect your readings by reading the buffer that your dna is in with and without edta added.

Hey guys,

Sorry for the delay, I was waiting to get the A320 readings but our QC section who uses the Nanodrop for primers won't take extracts in their clean room anymore.
So, we're trying to buy a second one for my section (research).
Maybe I can at least give them the tube with EDTA only. No risk of contamination here.

I don't like phenol-chloroform too much (like everyone I guess) because I loose the smallest DNA molecules. And since it's sometimes all I have. I can try with a good bone though.

Thanks a lot for the help. It's a really nice place here.

I wish everyone a good weekend,

Maddie