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DNA pellet disappeared after ethanol wash

DNA pellet ethanol wash

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3 replies to this topic

#1 mlk45



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Posted 01 October 2012 - 01:52 PM

I did a routine DNA extraction from tail snips. After precipitation w/isopropanol, I saw a white pellet in all of my tubes. I washed 1x with 70% ethanol and spun for 10 min per my lab's general protocol. When I looked at the tubes after the wash, the pellets in about half of the samples had disappeared! I couldn't even see a viscous-clear DNA pellet like I sometimes do; it was like there was only ethanol in the tube. I'm not sure what happened! Can anyone help?

#2 agorganic



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Posted 01 October 2012 - 03:03 PM

How many samples did this happen with?

Are you using a kit?

Try hold it up to the light to make sure the pellet hasn't got stuck to the tube (are you using mirofuge tubes?). I recently had something like this happen to me and I found it was usually in my technique, which meant I needed to critique my method having not done the process before.

I found the inverting the closed tubes sometimes meant the pellet got caught in the top of the microfuge tube. Are you vortexing? If so try not to let the supernatant reach the lid of tube. Also sometimes after centrifuging the pellet might get stuck to the tube and may be slightly different color between samples, even shrinking in size so that even holding up to light is difficult to find in the opaque tubes.

I had 24 samples and had to repeat the test 4 times, on the fourth test I added an extra 39 samples. Can't say for sure how many times this happened, but was one of the problems I encountered. Explain a little more of what your doing, as it may assist other also looking for questions and others with a bit more experience than myself. For instance, I used whole sheeps blood with a Qiagen Flexigene DNA kit.


#3 mlk45



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Posted 02 October 2012 - 06:09 AM

Thanks, Jesse. I don't use a kit. We put the tail snips in mouse lysis buff + proteinase k O/N at 60 degrees, add phenol/chloroform, vortex, spin, and then add isopropanol. Mix gently, spin, wash w/ethanol and let dry for 20 min. on heat block. This protocol comes from one of our former research associates who has been doing mouse work for at least 20 years, so I trust that the protocol is sound even though I may not be doing it correctly. After the wash, the pellet disappeared in 6 out of my 10 samples- I held up the microfuge tubes to the light but couldn't see anything in those 6.

I did leave the tails in the 60 degrees for longer than overnight- a full day- so I thought perhaps there would be no DNA but I got a nice pellet in all of them after the isopropanol, so I'm not sure what happened.

#4 Ameya P

Ameya P

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Posted 07 October 2012 - 10:33 PM

Its very likely that during the 70% ethanol step, the pellet was dislodged and stuck somewhere else on the micro centrifuge tube. For the 70% ethanol wash step, I make it point to visually check all my tubes for the DNA pellet. Try it the next time, you are extracting, it might give you the answer you are looking for.


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