Covaris recovery ratio

[pmiguel]

Not really damaged, just denature. Back in the early SOLiD days, there was a lot competition for genome coverage between ABI and Illumina. Seems like one consensus arrived at was that anything that heated DNA lead to worse coverage for extremely AT-rich regions. For what it is worth... I did not actually carefully read the resultant literature, so I can't say how well this claim is supported.

Anyway, around that time, ABI added a room-temp method for dissolving agarose for minelute (quiagen). They just added extra gel dissolving buffer (chaotrope.)

Since that point, I try to avoid high temp steps in genomic DNA library preps. But realistically it is not always possible. The zymo kit, aside from heating buffer, also recommends doing multiple elutions. So maybe you can get the same effect from multiple room temp elutions as you can from single high temp elutions?

--
Phillip

[razibus]

A temperature of only 60-70°C can dammage AT-rich DNA?

[pmiguel]

Thank you for your answer, it makes sense. Do you know that from your own experience or did you read a publication on the subject?

Back in the Sanger Sequencing days we frequently made libraries from sheared BAC DNA. Our yields from the gel were atrocious. We tried a bunch of different methods -- but it came down to anything that was silica based did not want to let go of the DNA if it was above 4 kb or so.

We went to a agarase-based method and that sucked too.

In the zymo kit, they recommend for larger DNA (>10 kb) to heat the elution buffer up to 60-70°C. They claim the kit works from 50 bp to 23 kb. Did you try zymo kit or only Qiagen kit?

Actually, I had not noticed that about the Zymo kit. We just tried it for the first time on some ~10 kb DNA cut out of a gel. this week Actually, I did not do the protocol myself so I don't know if the elution buffer was heated. I hope not, that can have detrimental effects on AT rich fragments.

Anyway, we did get DNA out of the slice, and the yield was pretty good.

--
Phillip

[razibus]

It's not a problem of quantitation. As I said "I'm measuring the DNA concentration with Qubit." Nevertheless, you have to check your DNA is properly sonicated by using the bioanalyzer.

Anyway, I agree with you : Qubit is the most reliable and convenient method to quantitate DNA.

[Isequencestuff]

we use the dsDNA Qubit kit (or Picogreen) to quantitate before and after Covaris shearing. We believe that this is the most accurate reading as previously compared to the nanodrop 1000 & Bioanalyzer. qPCR is accurate, but takes a lot more time to do than Qubit. In our experience both the nanodrop 1000 and the 2100 overestimate DNA concentrations.

[razibus]

Thank you for your answer, it makes sense. Do you know that from your own experience or did you read a publication on the subject?

In the zymo kit, they recommend for larger DNA (>10 kb) to heat the elution buffer up to 60-70°C. They claim the kit works from 50 bp to 23 kb. Did you try zymo kit or only Qiagen kit?

[pmiguel]

Thank you for your answer, it's very relevant.

I tried Qiagen columns but also Zymo Research columns. Both works perfectly on PCR amplicons but poorly on sonicated gDNA.

I will give a try to Ampure XP. Nevertheless, the elution volume is ~30 and 40µl, and I need only 10µl. (I don't like SpeedVac very much)

Anyway, I don't get why we lost so much gDNA in purification columns...

Once you get above a certain size, some clean up columns hold more tightly onto DNA and hence will not elute the DNA. We were previously unable to find any clean up column that would "let go" of DNA larger than 9 kb or so. This seems to be a characteristic of silica based methodologies.

Does Ampure have this issue? Not sure we have tested it, but probably not. Ampure gently binds DNA onto its magnetic beads via carboxyl groups in the presence of a precipitant (PEG).

--
Phillip

[razibus]

Thank you for your answer, it's very relevant.

I tried Qiagen columns but also Zymo Research columns. Both works perfectly on PCR amplicons but poorly on sonicated gDNA.

I will give a try to Ampure XP. Nevertheless, the elution volume is ~30 and 40µl, and I need only 10µl. (I don't like SpeedVac very much)

Anyway, I don't get why we lost so much gDNA in purification columns...

[addyblanch]

Just thought i'd add something from my experience. I too found this drop in quantity but found it not to be the fault of the covaris but of the qiagen kit.
After moving to a SPRI bead clean up the DNA is (more or less) the same before and after fragment.

[razibus]

I tried sonicating at 5kb and 3kb. The sonication profile looks good on the bioanalyzer.

However, the amount of DNA before sonication and after sonication/purification is very different. Indeed, the recovery ratio is not very good.

I'm measuring the DNA concentration with Qubit.

Do you, guys, have the same issue?

[suludana]

Counting Apples

Hi Floor121,

to count apples what do you prefer?
Put them into a machine accountant or count yourself one by one?
In the gel you are "counting" your DNA, in a nanodrop or Agilent 2100 is the machine that makes it, the machine is your eyes ...

[floor121]

Thans ECO.
Actually I fragment our sample following the protocol from SOLiD, and no glycerol has been used. So we can measure the concentraion before clean up.
I clean up our sample using Qiagen Qiaquick pcr purification kit, and the concentration before and after clean up is 70ng/ul and 20ng/ul (nanodrop), respectively. The concentration before clean up is great different as I descripted yesterday, but after clean up it is almost the same from nanodrop and Agilent 2100.
I also believe that the bioanalyzer should more accurate than nanodrop, but from the 2%agarose gel image the concentration of fragment DNA is more close to nanodrop????

[ECO]

The nanodrop number is subject to inflation based on other molecules in your sample (salts, free nucleotides, short oligos) (like any spectrophotometer).

How are you cleaning up your sample, and what is the concentration range you're in?

Overall, I have found the bioanalyzer much more accurate for quantitation prior to ePCR.

[floor121]

Hi,
Recently I am try to fragment genomic DNA by Covaris, and found something interesting. After DNA fragment I measure the concentration by nanodrop and Agilent 2100, the concentration from Agilent 2100 is only half of that from nanodrop???? Which is more convincing? Any suggestion about the results?