DNA pellet - Does not stick to the vial (Mar/09/2011 )
pito on Thu Mar 10 10:08:26 2011 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?
HomeBrew on Thu Mar 10 11:42:59 2011 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
gt_ameya on Thu Mar 10 12:53:46 2011 said:
pito on Thu Mar 10 10:08:26 2011 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.
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.
chimpsarehungry on Mon Mar 14 21:58:18 2011 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? I think that the problem is not that the "pellet" is not sticking to the tube, I think it's that the "pellet" is of low density and it floats. This makes me suspect that you actually have two "pellets" in your prep -- one DNA pellet which is pelleting at the bottom of the tube (and is likely not very visible), and another "pellet" of cell debris that is floating.
I'll bet you could fish this floating stuff out of the prep and proceed without it, and your PCRs would still work fine.
Probably, you have identified the problem HB.
I will try and look for the non-visible pellet, which makes the PCRs work and see, if I can discard the floating stuff, in a sample. The problem is that the phenomenon is quite inconsistent now. So, its difficult to predict where we will see floats and where we wont. Also, these aren't research samples which I could experiment with. At the end of the day, I have to give results with the samples I get.
but thanks anyways and I shall post new developments....
Do you want the pellet to stick just so you can get rid of the ethanol and then resuspend the pellet in TE? Because it does stick much better if you pipette the pellet and about a ml of ethanol out and put that into a 1.5ml tube and then centrifuge that for a few minutes then you can just pour off the ethanol and the rest will air dry quickly.
chimpsarehungry on Tue Apr 5 15:53:30 2011 said:
Do you want the pellet to stick just so you can get rid of the ethanol and then resuspend the pellet in TE? Because it does stick much better if you pipette the pellet and about a ml of ethanol out and put that into a 1.5ml tube and then centrifuge that for a few minutes then you can just pour off the ethanol and the rest will air dry quickly.
or I pipette out a little ethanol, so that i leave the pellet and ethanol in the same tube, spin it down and see if it sticks. Thanks for the advice, chimps
Actually, the person I was working with moved out of the lab and I have a new apprentice
. No sticky issues with his extraction (so far)!