miniprep: no precipitate after PEG treatment. - (Sep/14/2006 )
i wanted to post this long time ago, somehow always forgot to. we are doing ordinary miniprep here by alkaline lysis and the last step is PEG treatment after RNase. i can see the DNA in all steps but after i treat with PEG no precipitate. i actually go on and wash with ethanol and measure OD and check by gel and there is DNA. but sometimes there is no DNA . anyway it just feels very uncomfortable to go on with the procedure without seeing what I am extracting. any suggestions?
Don't PEG a miniprep!
It ain't worth the trouble, the extra time and worry. And by miniprep I am assuming a 2ml culture... or anything under 10ml. The extra removal of small nucleotude fragments isn't important for a DNA solution that dilute. It won'ty show on any difference on a gel.
I only do PEG percipitation on 100ml Midipreps and up.
thanx a lot! that is a reat relieve! however they are telling me here that PEG is necessary if i want to use the plasmid for ligation and transformation.
yes im only using 5 ml of culture.
Well, PEG percipitation is not absolutely nessary. PEG does make the DNA cleaner though and therefore the ligation work easier.
However if you gel purify the plasmid DNA after restriction digest, then the small fragments which PEG would normally removed are also removed. So no need for PEG if you gel purify.
(Gel purification also removes trace bacterial genomic DNA, denatured or uncut plasmid, RNA contamination, proteins, and keeps people in work at Sigma!)
My notes on PEG percipitation...
-Keeping the PEG DNA solution at minus 20 for 30min helps alot with the percipitation.
-PEG will not percipitate DNA if the concentration is too low or and if the DNA concentration is too high have badly reduced recovery rates. So for a give amount of DNA always scale the amount of PEG used.
-PEG is soluble in both water and ethanol.
-PEG contamination will cause serious problems with sequencing. It kills the reaction. One must wash DNA prepared via PEG very very throughly.
thanx a lot for your suggestions! since i cut out of the gel i'll skip the PEG step. i bet everyone in the lab will be super