14 replies to this topic

[Maddie]

Hi everyone

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 !!!

[HomeBrew]

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...

[skeuos]

HomeBrew, on Nov 5 2009, 06:11 PM, said:

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...

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.

[Maddie]

Hey guys,

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.
Have you?

[Maddie]

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.

[gogreen]

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

[Maddie]

Hi gogreen

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?

[mdfenko]

if your spectrophotometer readings are very low (low concentration) then your ratios are virtually meaningless.

[Maddie]

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.

[mdfenko]

Maddie, on Nov 6 2009, 03:11 PM, said:

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.

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)?

[chason]

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

[Maddie]

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?

[chason]

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

Edited by chason, 10 November 2009 - 01:04 PM.

[mdfenko]

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.

[Maddie]

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