18 replies to this topic

[Ameya P]

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?

[Adrian K]

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

[mdfenko]

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?

[Ameya P]

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?

[pito]

gt_ameya, on 09 March 2011 - 09:39 PM, said:

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?

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?

[Ameya P]

pito, on 09 March 2011 - 11:49 PM, said:

gt_ameya, on 09 March 2011 - 09:39 PM, said:

Should that make such a large difference?

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?

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????

[pito]

gt_ameya, on 10 March 2011 - 01:09 AM, said:

pito, on 09 March 2011 - 11:49 PM, said:

gt_ameya, on 09 March 2011 - 09:39 PM, said:

Should that make such a large difference?

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?

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????

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?

[Ameya P]

pito, on 10 March 2011 - 01:46 AM, said:

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

Today was day 3 in a row...

[pito]

gt_ameya, on 10 March 2011 - 02:04 AM, said:

pito, on 10 March 2011 - 01:46 AM, said:

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

Today was day 3 in a row...

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?

Edited by pito, 10 March 2011 - 02:11 AM.

[HomeBrew]

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?

[Ameya P]

pito, on 10 March 2011 - 02:08 AM, said:

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?

Its a medium sized pellet. I dint get the second part of your question.

Yes, I guess so... but my univ logo maintains the 'V' in 'rerum'. I dunno why? Do you speak latin? could you tell me whats the difference?

[Ameya P]

HomeBrew, on 10 March 2011 - 03:42 AM, said:

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?

Homebrew,

It is quite possible that the "pellet" has cell debris or something else. It is a bit sticky than when I isolate DNA. But it definitely has DNA. The PCRs work fine, irrespective of the nature of the pellet.

Its Qiagen Puregene Blood Core Kit B

[pito]

gt_ameya, on 10 March 2011 - 04:53 AM, said:

pito, on 10 March 2011 - 02:08 AM, said:

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?

Its a medium sized pellet. I dint get the second part of your question.

Yes, I guess so... but my univ logo maintains the 'V' in 'rerum'. I dunno why? Do you speak latin? could you tell me whats the difference?

With the second part of my question I ment: do you see a difference at the very beginning at the start of the protocol? Its possible he just doesnt have as many cells as you have to start with. This could explain why he has a lesser visible pellet.
But I think this isnt the case since you said it is a medium sized, very well visible pellet.

Weird thing anyway, if it all comes from the same samples and you use the same products.


About the rervm or rerum: in latin there is no "u" they dont know that letter. They use a "v" , but it can mean 2 things, depending on the word. In rerum it should mean a "u"  (or pronounced as a "u" (altough, the prononciation is "oe", but to state its oe we write u now..)
But in modern text they write the U in stead of the V , your university still uses the old style then thats why I got confused.
I think there are many universities with that logo.

[chimpsarehungry]

You can still fish it out of the ethanol with a pipette right? Try putting it into a different type of tube like a 1.5mL tube and spin that down and see if it sticks.

[Ameya P]

chimpsarehungry, on 14 March 2011 - 01:58 PM, said:

You can still fish it out of the ethanol with a pipette right? Try putting it into a different type of tube like a 1.5mL tube and spin that down and see if it sticks.

Thanks for your suggestion. Welcome to Bioforum as well    But could you tell me why should I do that? Do you think, its the tube that is responsible for the pellet not sticking?