1 reply to this topic

[zimmersk]

Hi,

This is my first post and I am desparately looking for some help recovering DNA from CsCl preps.  I have recently run into some problems precipitating DNA using sodium acetate (3M 1:30 dilution) and 2.5 volumes of ethanol.  I know the plasmid DNA is present as I remove it from tube post spin and then remove ethidium bromide with salt saturated butanol, I usually dialyse o/n in TE then recover the sample and complete the precipitation, but for some reason I have been unable to precipitate my DNA out of the last few samples I have run and I am not sure why, I know the DNA is there but after adding more and more ethanol the sample gets two large in volume and is cumbersome the handle, I attempted to to run the enitre sample over a qiagen maxi column and was able to recover some DNA but not at the concentration I am used to getting with the standard protocol. I also attempted to forgo the dialysis and add 3 vol H20 and 8 volumes ethanol and still nothing percipitated, even after incubation o/n at 4 degrees when I just added 1:10 volume sodium acetate and 2.5 volume ethanol a tremendous amount of salt precipitated, and now I dont know where the DNA is, if it is caught up in the salt precipitate or still in solution??? HELPPPP I really need this DNA and dont want to start another CsCL prep!!!

Edited by zimmersk, 14 June 2004 - 07:57 AM.

[zhongmindai]

zimmersk, on Jun 14 2004, 07:55 AM, said:

Hi,

This is my first post and I am desparately looking for some help recovering DNA from CsCl preps.  I have recently run into some problems precipitating DNA using sodium acetate (3M 1:30 dilution) and 2.5 volumes of ethanol.  I know the plasmid DNA is present as I remove it from tube post spin and then remove ethidium bromide with salt saturated butanol, I usually dialyse o/n in TE then recover the sample and complete the precipitation, but for some reason I have been unable to precipitate my DNA out of the last few samples I have run and I am not sure why, I know the DNA is there but after adding more and more ethanol the sample gets two large in volume and is cumbersome the handle, I attempted to to run the enitre sample over a qiagen maxi column and was able to recover some DNA but not at the concentration I am used to getting with the standard protocol. I also attempted to forgo the dialysis and add 3 vol H20 and 8 volumes ethanol and still nothing percipitated, even after incubation o/n at 4 degrees when I just added 1:10 volume sodium acetate and 2.5 volume ethanol a tremendous amount of salt precipitated, and now I dont know where the DNA is, if it is caught up in the salt precipitate or still in solution??? HELPPPP I really need this DNA and dont want to start another CsCL prep!!!

The DNA pellet in the tube is often invisible (said in molecular cloning: a labratory menual). I have experinced this before. Sometimes, the DNA pellet adhere to the tube wall and  if the DNA is not abundant enough, it will be invisible.
In my opinion, to pellet the DNA, you must mix the NaAc and ethanol in the tube well and centrifuge at >=12000 g for 10 min or more at 4 degree C, and transfer the supernatant to a new tube (Discard after ensure that the DNA has been recovered), 70 % ethanol wash and air dry, dissolve the DNA pellet (maybe invisible) in TE buffer or sterile deionized water, rinse the walls of the tube well with the buffer.
Wish this help you!

Zhong-Min Dai

Edited by zhongmindai, 14 June 2004 - 10:51 PM.