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ChIP - Is it possible to spin beads too hard? - (Jan/17/2008 )

All of the sudden I am not seeing any pellet during my IP? Is it possible to spin too hard and somehow destroy the beads so they no longer precipitate? I feel like I have heard or read that somewhere? If not then where did my beads go! smile.gif

-MKR-

QUOTE (MKR @ Jan 17 2008, 08:30 PM)
All of the sudden I am not seeing any pellet during my IP? Is it possible to spin too hard and somehow destroy the beads so they no longer precipitate? I feel like I have heard or read that somewhere? If not then where did my beads go! smile.gif


What speed are you spinning? From my knowledge beads should be spun very fast. I use 1500rpm.

-Clare-

Well I started at 1500, then 3000, then our max 13.2krpm. Still seeing nothing???

They've got to be there right? I have placed my samples in the freezer and will spin them again (maybe the agarose is melted?). I am at a loss.

QUOTE (Clare @ Jan 17 2008, 01:13 PM)
QUOTE (MKR @ Jan 17 2008, 08:30 PM)
All of the sudden I am not seeing any pellet during my IP? Is it possible to spin too hard and somehow destroy the beads so they no longer precipitate? I feel like I have heard or read that somewhere? If not then where did my beads go! smile.gif


What speed are you spinning? From my knowledge beads should be spun very fast. I use 1500rpm.

-MKR-

QUOTE (MKR @ Jan 18 2008, 12:54 AM)
Well I started at 1500, then 3000, then our max 13.2krpm. Still seeing nothing???

They've got to be there right? I have placed my samples in the freezer and will spin them again (maybe the agarose is melted?). I am at a loss.

QUOTE (Clare @ Jan 17 2008, 01:13 PM)
QUOTE (MKR @ Jan 17 2008, 08:30 PM)
All of the sudden I am not seeing any pellet during my IP? Is it possible to spin too hard and somehow destroy the beads so they no longer precipitate? I feel like I have heard or read that somewhere? If not then where did my beads go! smile.gif


What speed are you spinning? From my knowledge beads should be spun very fast. I use 1500rpm.



13.2Krpm is way too fast! Are you sure you mixed your beads properly before adding them to your sample? (ie: maybe the beads settled and you only added the liquid portion of the 50% slurry?)

-Clare-

Hi everyone, I spin down the agarose beads at 4000 rpm at the washes and at 13000 rpm with the elution steps and I see the pellet very well; I think that maybe you aren't mixing well the beads (as Clare wrote), you can even try to cut the end of the tip to ensure you that you are pippeting the beads correctly.

I wish you luck!!!

-ribonucleico-

I saw the bead pellet when I washed the beads, all I did was add the sample, mix a little, and spin. Now I can't seem to see anything?

EDIT: What do people think is the best type of tube to use. I use the normal 1.5mL eppendorf tubes. ut I vaguely remember in the past seeing some other brand/type of tube that was all clear (no lines or cloudiness from thicker plastic at the bottom). That may help in seeing my invisible pellets.

QUOTE (ribonucleico @ Jan 18 2008, 02:46 AM)
Hi everyone, I spin down the agarose beads at 4000 rpm at the washes and at 13000 rpm with the elution steps and I see the pellet very well; I think that maybe you aren't mixing well the beads (as Clare wrote), you can even try to cut the end of the tip to ensure you that you are pippeting the beads correctly.

I wish you luck!!!

-MKR-

Hi, I normally use 2 ml eppendorf tubes, so I donĀ“t think that's your problem. One question in what moment during the procedure are you exactly "losing" the pellet??.

-ribonucleico-

Hi there, I don't think the tubes is the problem too. Most likely you didnt mix the beads properly. Also, I think that 13.2K is too fast for spinning beads, but that depends on which step you're at too.

-janita219-

Hi, there is a possibility that the speed is way too high for the beads. The specifications for the beads usage should have stated the range of centrifugation speed. For the beads Im using, it's 800rpm tp 1.2krpm.

Hope this helps..

-jae.tan-