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Midiprep - (Jun/11/2007 )

Dear all,
Could you please help me regarding my midiprep as I keep losing the DNA, could any one tell me the best way to preserve the DNA pellet.
Thank you in advance

-Noha13-

Could you please elaborate more? How did you actually lost your DNA pellet?? Did you dry it in vacuum dryer? If yes, I would suggest you not to use that. Normally, I just air-dry my DNA pellet upside down on a paper towel for about 30 minutes or until I have finished my other works (For your information, I am multi-tasking smile.gif ). A little bit of EtOH left in your tube will not have any significant effect to your experiment.

-virus_fan-

After adding propanol and centrifugation I remove the supernatent and I cannot see the pellet and then I add the ethOH and centrifuge and I leave it to air dry up to 30min and i still have the alcohol in it, but I then add water 300ul and put it in a new tube so is this right and if so, why I lose my DNA??

-Noha13-

QUOTE (Noha13 @ Jun 11 2007, 02:56 PM)
After adding propanol and centrifugation I remove the supernatent and I cannot see the pellet and then I add the ethOH and centrifuge and I leave it to air dry up to 30min and i still have the alcohol in it, but I then add water 300ul and put it in a new tube so is this right and if so, why I lose my DNA??


It sounds alright to me. But what is the speed you used to spin your sample? For my practice, I spin it at 13000rpm for 5 minutes.
I would think that by adding 300ul to your sample is a little too much. 100uL of water should be sufficient.

-virus_fan-

Decant the isopropanol into a clean 50 ml conical so that in case your pellet is getting dislodged, you can always retrieve it. This can happen quite frequently. I spin it as fast as the centrifuge allows me to so that I can see a pellet and also that it does not dislodge.

Good Luck !!!

-scolix-

thank you
For the last two steps I use 5000g for 1hour which is mentioned in the qiagen manual, and I will try to resuspend the pellet. But is there any way that I could see the pellet.

-Noha13-

How do you see the pellet, how does it look because I read that it is glassy.

-Noha13-

For the isopropanol step, I centrifuge it as fast as possible, like 15,000 x g. This way the pellet is still attached to the wall of the tube.

True, DNA is colorless, but there might be some salt which might give it the white appearance. Even lots of colorless DNA in a single spot could give you a film of DNA on the walls of the tube, which can be seen.

-scolix-

QUOTE (Noha13 @ Jun 13 2007, 06:05 PM)
thank you
For the last two steps I use 5000g for 1hour which is mentioned in the qiagen manual, and I will try to resuspend the pellet. But is there any way that I could see the pellet.


Huh? I thought after elution, add isopropanol, the recommendation in Qiagen for centrifuge speed is >20000g? For midiprep, with high copy plasmid, I normally use 200ml culture (I know Qiagen recommend much less, but I found it work for me), and always see DNA pellet. In the few cases that I did not, I did something wrong and my midiprep was essentially lost.

-Almasy-

After centrifuguin for an hour at 5000 (max speed on a big centrifuge) i centrifuge again in an eppi centrifugue at max speed and recover A LOT!.. 40% OF THE TOTAL YIELd is from dna not precipitated in the first round

QUOTE (Noha13 @ Jun 13 2007, 12:05 PM)
thank you
For the last two steps I use 5000g for 1hour which is mentioned in the qiagen manual, and I will try to resuspend the pellet. But is there any way that I could see the pellet.

-tertu-