trouble resuspending DNA after EtOH ppt'n - (Apr/14/2005 )

Hello all,

I need your help. I precipitated DNA using sodium acetate and ethanol and I got a nice pellet.

I used buffer EB from Qiagen kits to resuspend it (Tris, pH 8.5) but not all of the pellet went back into solution.

How do I get it all back into solution??

I did a spec analysis of the solution and it gave me 0.667 ug/uL.

My DNA is a plasmid 18kB in size.

Thanks.

Has your DNA been overdried? I also use EB buffer a lot and have never had this problem.

did you perform a wash step after your EtOH precipitation?

what was your A260/A280 ratio?

Could it be excess salts that are not going into solution?


N

I dried it on a 37C heatblock for <30 mins, just until most of the big water droplets are gone. Then added buffer EB. And yes, I washed the pellet with 70% EtOH after precipitation.

It's nothing crucial, as I got enough of the solution for the concentration to not matter. Just wondering where I went wrong.

Perhaps I should've resuspended the original maxiprep DNA in water?

hi...

regarding ur problem of trying to completly dissolve the DNA...

try the following

use 10 mM Tris HCL (pH 7.6) and 1 mM EDTA (pH 8.0) buffer....to dissolve the DNA and then keep at 65 degree temp for abt 15mins.......

also refer an article....Nucleic acid research, Vol 19, No 19 page 5444..
(Debomoy Lahiri and J Numberger Jr)

hope this shud solve ur problem....lemme know if it worked...

all the best.....

best wishes
swarup

Hello all,

I need your help. I precipitated DNA using sodium acetate and ethanol and I got a nice pellet.

I used buffer EB from Qiagen kits to resuspend it (Tris, pH 8.5) but not all of the pellet went back into solution.

How do I get it all back into solution?? 

I did a spec analysis of the solution and it gave me 0.667 ug/uL.

My DNA is a plasmid 18kB in size.

Thanks.

thanks for the reference, I'll see if I can dig up a copy of the journal in our library.

hi
is there a possibility RNA remains in your prep?
i mean try to heat your sample on EB at 60° 10' and if you get a pellet back it's probably RNA

As of the moment, I'm thinking it's just too much salt. I'm doing more maxipreps and I'm going to be more careful this time.

swarup: I sent you a PM re: the reference. thanks again.