I extracted DNA using extraction column. It appeared that there was a lot of cell debris in the extract. Lot of impurities appeared in the nanodrop specking. How can I remove the cellular debris without repeating the extracrion and loosing reagents?
Does centrifugation helps?
Hi there. If you don't want to re purify ethanol precipitation followed by spinning and then washing your pellet with 70% ethanol could help. Take your eluted sample add 3 vol of ethanol plus 1/10 vol 3M Sodium acetate pH 4-5. Incubate for 1 hour at -20C. Spin at 13k for 15-20 min. Remove most of supernatant. Wash pellet in 70% ethanol by adding 500ul. Vortex briefly. Spin again for 5 min. Remove most of supernatant taking care not to dislodge pellet. I will do this by using a p200 tip to remove most and then a p10 tip at very bottom. Spin for 1 more min to bring down residual ethanol on sides of tube. Remove this (
Thanks i appreciate your help.
Do I have to centrifuge the eluted DNA first to get rid of the cellular debris?
Hi Emile. Is this stuff that was eluted from your spin column; that is what was eluted was aqueous and cloudy and this insoluble matter has precipitated out since elution. Just trying to clarify exactly where this came from. To be honest if as I suggested that would be very unusual and might imply a sub optimal purification. Ethanol precipitation could help but if I experienced a purification like that in all honesty I would perform again. Can I ask you when you initially re suspended your pellet in lysis solution did you make sure it was totally lysis ? Also what is your DNA plasmid or genomic DNA. I am just trying to ascertain based on specifics and type of DNA what advice would be best suited to your situation. If you like I can send you specific SOPs for plasmid extraction and genomic purification. I have performed much of both over the years and have optimised or improvised on the standard kit protocols for more consistent results
Talk soon
Hi Emile. One more thing for now. If you are absolutely sure it is just cellular debris them yes spin for 5 min @ 6k then ethanol precipitate and wash as already described. For advice for next time down the line contact me for SOPs. In the mean time I would be interested to know if you were extracting plasmid or genomic DNA and which kit you used and at what stage debris appeared
Thank you so much Dr. Hall.
I am pretty sure that the impurities are cellular debris because of a protocol gap in the extraction process. I guess that the DNA was not well washed with the buffer and the debris were eluted with the elution buffer. The extracted DNA is genomic. I am using the Qiaamp DNA extraction kit from Qiagen.
I will try the centrifugation and the ethanol precipitation tomorrow and give a feedback.
Hi emile,
An old method you can follow by running the crude DNA in the gel and after completion
the DNA can be eluted from the gel.... But agreed with Lawrence
Regards
Aamir
Hi Dr. Laurence,
I did the recovery method that you suggested and I was able to recover 4 samples out of 9 which is great. I got pretty good DNA concentration.
Thank you.
Hi Emile
that's great: I would add 1 more thing. If recovery of DNA is an issue you can add 1ul of 20mg/ml glycogen as a carrier. Add this once you have added your sodium acetate and before you mix in your ethanol. This will greatly improve your recovery - especially if over all DNA yield just a few ug - and the other advantage of adding glycogen is that the pellet is very white and conspicuous until dry and so you can manipulate with confidence and not worry about losing. The carrier by definition incidentally is inert so does not interfere with down stream reactions. I have done this a lot. Recovery is invariably in excess of 90%. So do as as I stated as you have done before plus add glycogen then precipitate as already described. Find attached some info. As long as the glycogen is molecular grade and 1mg/ml to 20mg/ml just add 1ul. You can buy from Sigma Roche or Fisher (as attached)
https://www.lifetechnologies.com/order/catalog/product/R0551
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