2
$\begingroup$

I am having trouble resuspending purified human genomic DNA. The genomic DNA is purified using phenol:chlorofom from ~ 1 mL of blood. After ethanol precipitation, I normally see a large pellet which is then resuspended in 150 uL of TE (10 mM Tris pH 8.0, 1 mM EDTA).

But the resulting DNA solution is extremely sticky. There are the options I have tried:

1) Incubation at 37C for 1 hr to overnight

2) Incubation at 50C for 1 hr to overnight

3) Incubation at 65C for 1 hr to overnight

The 65C incubations for up to 3 hours do make the DNA solution less sticky, but in the mean times, cause increasing DNA smearing when running on agarose gel. After overnight incubation at 65C, the DNA solution is no longer sticky but shows extensive smearing on gel, indicating DNA degredation/fragmentation at high temperature.

I have also tried resuspending the DNA in 8 mM NaOH, then neutralise it with Hepes, but this doesn't help either.

I am thinking to try silica/glassmilk beads based methodology. Will genomic DNA captured in silica beads be easier to elute and resuspended without the "sticky" issue.

$\endgroup$
  • 1
    $\begingroup$ Sounds like an impurity in your prep as opposed to they way you're resuspending. If silica worked it wouldn't be because solubilization of the DNA was easier, but rather because impurities were more efficiently removed. It's not like if you have completely pure DNA on a column vs. in a pellet the pellet could be more sticky upon solubilization; the end product of dissolved DNA would be identical. Have you checked the OD260/280 of the sticky stuff? Or, maybe the DNA is just too concentrated; maybe try more solvent? Some people on the internet are saying they have this problem from over drying. $\endgroup$ – Jory Apr 3 '16 at 6:43
  • $\begingroup$ Purified DNA have a good 260/280 (~1.85) and a nice graph (nanodrop). I don't think the DNA is over dried, as soon as it's washed in the 70% ethanol, I aspirate all the alcohol and put TE into the bottom of a tube immediately. $\endgroup$ – Green Apr 3 '16 at 7:58
  • 1
    $\begingroup$ When you say 'sticky' do you mean viscous, a solution with high viscosity? That is precisely what you would expect, or predict, for purification of intact, high molecular weight, genomic DNA. Your own data reveals this to be the case, as assayed by agarose gel electrophoresis, when the viscosity of the solution decreases the DNA fragments in the tube get shorter. It follows that longer fragments of DNA are likely causing the increase in viscosity. The question is, what do you need to do with the DNA? $\endgroup$ – mdperry Apr 4 '16 at 1:19
  • $\begingroup$ What about proteinase K? You mentioned the phenol/CHCl3 extraction, but that is typically preceded by a digestion with proteinase K at 65 C in the presence of SDS and EDTA. $\endgroup$ – mdperry Apr 4 '16 at 1:22
  • $\begingroup$ 'Viscous' in the word. I tried with and without proteinase K and noticed that proteinase K can be omitted without affecting the quantity nor quality (absorption spectrum). The DNA is for next gen sequencing. It's true that DNA will be fragmented in most sequencing platforms. But we want to keep the DNA as long as possible for something like pacbio which requires long intact DNA $\endgroup$ – Green Apr 4 '16 at 2:48
1
$\begingroup$

Just a thought, have you tried eluting the DNA in Molecular Grade Water instead of TE?

  1. For DNA which are supposed to be used in downstream applications like sequencing or transformation, elution with Water instead of TE buffer is frequently done.

  2. I agree longer genomic DNA can be sticky upon elution, but if by chance the DNA is not entirely dissolved when you add the eluant (you see some very small clumps of flakes instead of a homogeneous clear solution) , that may be because of the presence of low amounts of alcohol (organic solvent). That's why it is recommended that after aspirating the alcohol, you should leave the tube opened for ~10 minutes for the trace amounts of alcohol to dry out before you add the eluant.

Sorry if you have done these things already, just wanted to be sure.

Hope this helps!

$\endgroup$
  • $\begingroup$ It's true that water is sometimes recommended for elution. But in general TE is better for (1) resuspension as the alkaline pH is better at "solubilising" DNA and that the pH of water can be fluctuating between 5 ~ 7; (2) stability as the EDTA prevent extraneous nuclease, Qiagen has a related note $\endgroup$ – Green Apr 4 '16 at 4:18

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.