Qubit vs gel - results seem off

[pandrade]

Hi everyone,

Lately I have been extracting DNA to do genotyping-by-sequencing, and one of the requirements of the company that is receiving the samples is that DNA is at a minimum concentration of 50 ng/ul. After a first look at the gel, I was sure I had recovered a lot of DNA, but after quantifying with Qubit (dsDNA, broad range) I get results that are below 50 ng/ul for most of the samples. I tried using different Qubit kits or different incubation times, and the results are very similar. Other colleagues have looked at the gel and the readings and agreed that the results are odd.

I attach an example of part of the samples. Most of them seem good (they have high molecular weight) but the readings are mostly from 20-40 ng/ul.

Most of these samples had very low readings for NanoDrop ratios, indicating probable protein contamination, but I thought that it would not be a problem for the Qubit. Can an excess in contaminants impede the procedure, or do the samples actually have low DNA yield?

[kerplunk412]

I believe the Qubit manual has a section that mentions many contaminants specifically, so that is a good place to start. Also, it would help if you could share some more details, such as how the DNA was purified and a little better description of the gel image (ladder, amount loaded?). I think it would also help if you could post the actual Nanodrop traces, those can be pretty useful.

[nucacidhunter]

Hi everyone,

Lately I have been extracting DNA to do genotyping-by-sequencing, and one of the requirements of the company that is receiving the samples is that DNA is at a minimum concentration of 50 ng/ul. After a first look at the gel, I was sure I had recovered a lot of DNA, but after quantifying with Qubit (dsDNA, broad range) I get results that are below 50 ng/ul for most of the samples. I tried using different Qubit kits or different incubation times, and the results are very similar. Other colleagues have looked at the gel and the readings and agreed that the results are odd.

I attach an example of part of the samples. Most of them seem good (they have high molecular weight) but the readings are mostly from 20-40 ng/ul.

Most of these samples had very low readings for NanoDrop ratios, indicating probable protein contamination, but I thought that it would not be a problem for the Qubit.

If your gel image is from top row of a multirow gel, they would benefit from a clean up with a column or bead for GBS. Clean up would remove possible contaminants, viscous material and also you can adjust (concentrate) concentrations by elution volume. Also I would look for possible denaturation during extraction process because Qubit measures dsDNA portion not ssDNA which would not be evident in gel.

[pandrade]

Hi guys, thank you for the reply. I attach a picture of a gel (0,8%) of 8 of those samples, with their concentrations measured with Qubit BR. The ladder is a HindIII lambda DNA ladder, so the samples are mostly genomic DNA. I also attach a picture with two of the readings on Nanodrop, so you can see the type of trace and the ratios.

Edit: Oh, and I have tried cleaning with magnetic beads, but that didn't change the nanodrop readings. What could cause much denaturation of dsDNA into ssDNA?

[SNPsaurus]

How much are you loading onto the gel? It looks like a lot of good DNA to me (although 50 ng/ul sounds like a high required concentration to me as well). The Qubit measurements seem to be pretty consistent with the gel images as well, which is a good sign.

If you have a lot of volume to work with, just dry them all down a bit, measure, and dilute to 50 ng/ul. If you are really worried about ssDNA, test a sample with a ssDNA nuclease against some known dsDNA and denatured DNA (ssDNA) and see which your sample resembles more.

[IdahoRAD]

I think that a clean up is a good idea also. And then elute them in a small volume, quantify, then dilute to 50 ng/uL. But I also agree that 50 ng/uL seems pretty high!