desalting/concentrating DNA - (Mar/22/2006 )
Hi guys,
would you recommend a kit/protocol for desalting/concentrating small amount of DNA? (Sick of percipitation with glycogen ...) Thanks.
you can do regular isopropanol or ethanol precipitation:
http://epsc.wustl.edu/~yata/Untitled-2.html
I use Qiagen's PCR clean-up kit.
instead of isopropanol or ethanol, i would use Butanol which actually pellets better.
Add 4vol of butanol vortex30'' and spin 5' full speed. Wash with 70% etoh.
You'll get better recovery than by etoh or iproh
thanks for the replies.
i have a couple comments:
using EtoH or IsopOH results in a lot of loss, at least in my hand.
for Qiangen PCR cleanup kits: usually they are okay, but sometimes they screwed me up completely.
One more question, sometimes I use glycogen for percipitation. However, it seems to mess up my Nanodrop reading after I resuspend the sample.
Fred33,
How much DNA is recovered after butanol precip., compared to the DNA concentration before?
I did 2 different precipitations on 1 sample with 180ng/ul DNA, one with ethanol and another with propanol. WIth isopropanol I had 210ng/ul after precipitation, and with ethanol - 200. This is not a significant difference!
How concentrated the DNA is after precipitation usually?
THanks!
Add 4vol of butanol vortex30'' and spin 5' full speed. Wash with 70% etoh.
You'll get better recovery than by etoh or iproh
Bu usually i've noticed that by EtOH precip, i loss 15% of my sample
With butanol, i loss less material.
I will do an assay based on 1µg if you want, but i've not time currently.
So it's for tomorrow.
anyway, with EtOH precip, it's recommended to store the tube 20-30' at -80 to enhance DNA clumping. You can spin directly but it's known that drives loss of small fragments.
With IprOH, this step is avoided because it's more hydrophobic (1:1 v/v versus 1:2.5 v/v)
Butanol can dry your pellet very good but needs good washes as it doesn't accept salts (EtOH do accept them as it's diluted by your sample, and IprOH too).
it doesn't accept salts
it's also a reason why it precipitates better nucleic acids.
I see so many different precipitation protocols, that now I don't know which one is correct!
With Ipr. I saw that -80C for 30' is required.. sometimes I see that with EtOh only -20C for 15 min is required..hmm, 
I care because I don't have so much sample so I can try many different precipitations with it..
Thanks for suggestions!
you can read this thread for iproh and etoh :
http://www.protocol-online.org/forums/inde...wtopic=3874&hl=
that one is for "tips on etoh precipitation of nucleic acids"
http://www.protocol-online.org/forums/inde...wtopic=5564&hl=