A260/A280 at 1.3 - HELP (Nov/05/2009 )
I extract DNA from bones and I recentely tested the A260/A280 knowing it had to be above 1.8.
I did not expect 1.3. All 12 extracts I have have the same ratio. I had digested with proK and cleaned-up with the Qiagen MinElute kit, so what can I do next? Double Qiagen clean-up doesn't help.
Please, please help me !!!
First off -- welcome to the BioForums, Maddie!
Now, what do you intend to use the DNA for?
Personally, I don't ever get hung up on A260/A280 ratios (as many people around here have probably heard me speak of before), and would certainly not let a "bad" one stop me from continuing an experiment. Our lab doesn't even own a spectrophotometer, and I haven't looked at an A260/A280 ratio in years, and it's never impacted my success rate...
HomeBrew on Nov 5 2009, 06:11 PM said:
I'll ditto that. I've often had bad (1.4-1.6) ratios for RNA that looked perfectly fine on a gel, and worked great for reverse transcription. Sometimes it also depends on the spec - one spec in the lab will always give me a bad ratio, while another one gives a rather nice ratio.
The need for purity does depend on the downstream application.
Thanks a lot for the nice Welcome
I've been dreaming of a place like this for so long. I feel like I finally meet people who talk my language :-)
I use my DNA for PCR and qPCR. I decided to look closer to DNA purity because I noticed that extracting more bone wouldn't result in better DNA yields. Ex: 1g of bone won't give me anything close to 10 times what I get with 100mg. I know it's not an exact Science but still, I would expect to see more than a x4 increase. So I wondered if the DNA purity could be at fault. I extract small amount of bone in a small volume that I finally concentrate to 100ul but big amounts of bone extracts also end up being concentrated to 100ul. I've looked at the proK, at inhibitors, but the concentration of all these components should be the same in the initial extraction buffer whatever the amount of sample (I increase everything proportionally).
I'm at loss.
Some colleague who work on soft tissue also noticed that it was better to use small amounts. I haven't found anything in the literature about this.
It's funny how I always get a big silence after I ask this question
Please, bring me a nice big wall, so I can bang my head on it.
Hey Maddie, Why don't you decide the amount of starting material according to the requirement of DNA/RNA for your downstream applications and stop wondering and bothering about different starting materials giving different yields! there can be a lot of factors like improper homogenization resulting lower yields, losses during purification, blah, blah, blah....
In my experience also, the low 260/280 and 260/230 ratios of RNA/DNA had not given me troubles in for qPCR and microarrays
I think I should have mentioned that I work in the Forensics with very VERY bad bones. I need to send extracts to a company for next generation sequencing (don't know which one yet) that will sequence it for me but all I see is ratio A260/A280 must be between 1.8 and 2 and the amount of DNA they require ..In my world, ug doesn't exist. We talk, at best in picograms.
Also, increasing the DNA yield can make a huge difference in getting a DNA fingerprint with 17 loci instead of 3.
I really need to increase the DNA concentration (without increasind inhibitors and/or proteins as well). I pooled as much as I could but this is very limited in term of increase.
For people who extract DNA from soft tissue: how much sample do you start with? Have you ever tried to extract 100 times more?
if your spectrophotometer readings are very low (low concentration) then your ratios are virtually meaningless.
They are between 0.25 and 0.65. Is that too low?If I multiply by 50, it gives me "what I call" huge amounts of DNA, but again maybe my specialty biaised my thinking about what is low and what isn't.
Maddie on Nov 6 2009, 03:11 PM said:
no, the readings are okay, not too low (i thought it may be low because you wrote about low yields). then there is another reason for the low ratio.
are you sure that you didn't carry over anything else from bone (eg calcium compounds)?