Hey Rima that white fragment might be DNA or protein you can check contamination by taking O.D. at 280nm wavelenght as A.A. TRP and TYR will absorb at that wavelenght.
All d best
I am going to do a lot of assuming here, but if you are doing a ChIP and have lysed the cells with a buffer containing SDS, this is the SDS becoming cold and precipitating. Then as the solution gets warmer during centrifugation it gets re-dissolved. But you can try it for yourself - make a mock reaction without the DNA, put it on the ice together with your samples and check. If this is the case, you should be able to make the crystals disappear by rubbing the tube in your hands to warm it up.
Oh, and in that case you don't need to "troubleshoot" since you are purifying the DNA anyway to remove all other things - SDS among them.
Could likely be DNA or chemical in solution precipitating out, DNA is normally threadlike and precipitates easily, redissolve and check wavelength, simple test or electrophoresis.
Hi thanks all for your reply...I had pure dna (from Sigma aldrich) and I did not use SDS...I just dissolved the dna in Tris-HCl buffer...no EDTA either...and am gonna use the sonicated dna for viscosity measurement and not for ChiP...the fragments are not redissolving...they just keep floating:(
This is weird... but do try the no-DNA sample to see if you still get the precipitate. Other thoughts: Make sure that both the DNA and the buffer concentrations are correct and that you are not over-saturating them (which would cause precipitation - then again, this shouldn't be floating after centrifugation). Another thing is to make sure that the sonicator probe is clean (I've seen filthy tips left from previous users before)... If you can't seem to fix this or figure out what it is, try collecting the floating pieces and check by electrophoresis if they are DNA or not. In the end, do what we always do when we can't find an explanation: try new solutions of everything. Do tell us what you come up with, I'm really curious now...
Hi I tried with no dna and it gave white floating particles though not to the same extent as with dna. I ethanol precipitated the dna solution containing fragments followed by centrifugation but the particles continue to float...so right now the guess is the particles are not from dna...may be theyt are comin from the tarson falcon tubes due to sonication fro long time...can i use glass tubes for sonication?or may be i should change tubes every 5mins of sonication...
If recovery of sample is not a problem i.e. expendable then you may use glass.
There's two main types of falcon tubes, the clear glass-looking ones and the mat, semi-transparent ones. You could just try a different type. Or even use FACS tubes if you have any. Though I somehow doubt it's the plastic (then again, you could do an even more negative control and only put water in a tube). Generally, if the particles aren't your DNA, and they don't contaminate your sample (since they don't precipitate), I wouldn't be bothered much. Regarding your specific question, I'm not sure how well glass tolerates ultrasound, maybe check your ultrasound generator's manual?
I would be checking viscosity with this sonicated dna...using a microcapillary viscometer in which these particles would affect the readings...i altered my protocol and am gin=ving real long cooling breaks between the pulses...also am transferring the sample from time to time to fresh tubes...yet the particles come...although in much smaller quantities now...im using the semi transparent falcons...may be should change the tube or find some way of removing the particles...
Try to measure OD at 260 and 280 before and after the treatment to see if total DNA amount is decreased as if this was the case the concentration of the original DNA in the solution should be reduced.
Rima,
it seems like your sonicator probe is the sourse of this white powder. Clean the probe well or change it for a new one. At normal DNA concentrations, DNA should not be behaving this way. Since you see the white floating material in samples without DNA, it is definitely a sonicator problem.
Sonication could cause protein denature which could show "floating material" although in most cases it can be settled down with high speed centrifugation. I don't believe the probe was the sourse of the problem as it seems "too easy" and it must be washed before and after uses as a rountine or basic lab procedue.
If you are dissolving the DNA from a lyophilized pellet, the flakes may be undissoved DNA. More likely if the DNA is higher MW. Try heating the sample to dissolve the DNA.
White thready or spindly particles which precipitate out is the DNA, what you tried and got some particles may just be contaminant., try doing an electrophoresis of that to separate grain from chaff.
I had a similar problem in a different experiment (nucleic acids are not my topic): I had a plasmid dispersed in a water solution that "precipitated" (a white and visible material floating in the buffer was formed) after just to contact with a liposome dispersion with a small % of positive charge (cationic lipid). I supposed that the cationic character of the liposomes affected the dispersion of the plasmid, perhaps because it is necessary to fit well the % of positive charge with the amount of plasmid present.
If you use a titanium probe sonication in your process, could it be that some of the material becomes detached from the probe and this will affect the solubility of your DNA?
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology