DNA pellet - Does not stick to the vial (Mar/09/2011 )

This is just an observation.

Although, we use the same DNA extraction kit, and process the samples from the same source, I have observed that when a lab mate of mine processes samples, after 70% ethanol wash, the DNA pellet refuses to stick to the tube. Usually, we centrifuge for a 1min but even after centrifugation for 3 minutes, this pellet stays in 70% ethanol.

This makes supernatant discarding quite tough and time consuming. But what bothers me the most, is why isn't the pellet sticking to the tube?

Hi Ameya,
Do you mean you saw the pellet, but it doesn't stay there?

do you use everything the same?

maybe your labmate uses a different brand of tube or a different model (eg- siliconized)?

or a different (older) bottle of 70% ethanol?

Yes Adrian, the pellet is seen, but refuses to stick to the tube.

mdfenko, NOTHING is different. The lab mate is new to the lab. So, everything being used is mine.
Its just a different pair of hands doing the job.

Should that make such a large difference?

gt_ameya on Thu Mar 10 05:39:27 2011 said:

Yes, it could make a difference.

Did you see him do it?

And you do see the pellet? Its not that you dont see a pellet (or hardly at all) when the labmate does it?

pito on Thu Mar 10 07:49:12 2011 said:

If you say, it could make a difference, could you please explain how?

The whole process is done under my supervision. The ingredients used are exactly the same.

And as I said, I am not worried about the pellet not being there.... I am worried about the pellet not sticking to the vial. How do you explain that????

gt_ameya on Thu Mar 10 09:09:34 2011 said:

Give a protocol to 20 people and give them the same substances (same bottles etc) and you will get 10 different results in the end...
There are always factors influencing the results: shaking too long, shaking to hard, doing something different when extracting.. Always hard to tell.
Its also possible that it all starts at the beginning: the DNA extraction itself, when you add the cells to your tubes.
Let him to the first step and then you take over and let him then work with your first step.
And see what the results then are. Maybe he just doesnt take enough DNA at the start (not enough cells or something like that).
But in your case: if you indeed see (supervise) what he does, it is indeed a bit strange that you get such a different result.

And why it doesnt stick to the vial.. hard to tell. It is indeed weird.
But you do see a pellet, right? Or not? Because this makes a differce.

BTW: how many times have you observed this "not sticking" to the vial? Only once? Or is it everytime?

pito on Thu Mar 10 09:46:34 2011 said:

Today was day 3 in a row...

gt_ameya on Thu Mar 10 10:04:43 2011 said:

Oh ok, its not a fluke then.

It does stay strange.

Do you see a big pellet that doesnt stick to the vial or rather a very small one?
And do you see a difference in pellet right after you started (when you did the extraction, gathered the cells itself)


PS. What does rervm cognoscere cavsas means? Shouldnt it be: rerum cognoscere cavsas?

Fish the floating pellet out with a pipette tip and see that it is DNA. It's likely that the "pellet" you're seeing is not the DNA, but some polysaccharide or other cell debris.

What protocol (kit) are you using?