dna purification - (Sep/13/2006 )
does anyone have any recommendations for a reliable and high-yield method of dna purification? we're looking for ways to purify both genomic dna and fragments rangind from 200 b.p. to 2 kb.
so far, we've used zymo's clean and concentrator kit (a column-based purification method) as well as tried a couple different things with qiagen reagents and columns, but we can't get anything higher than a 20% recovery (and yes, we've tried eluting in water and TE (pH 8 and 9) and heating to 55C for 5-10 minutes. So far, that's the best we can get..
help please! 
very strange, I hear good things about Zymo kits. I use qiagen PCR clean up column on a regular basis (it is the labs only kit, we do everything ourselves) and never had any problems.
So risking sounding silly, there are three point which I want to note,
The ratio of Q buffer volume to gel size must be correct. Too much gel, and the Q buffer will be too diluted to disrupt and dehydrate the water structure surround the DNA molecule. WItout proper disruption, the electrostatic attraction towards silicon within the column will not be strong enough to pull out the DNA. Making the silicon binding column as useful as a filter paper.
Adding isopropanol help. (It dehydrate the water structure)
and finally, the columns have a maximum binding capacity. Officially of 10 micrograms but my lab collegue has worked that to be around 15 micrograms. Anything more, and it'll just wash through with the solution.
I currently use Qbiogen's GeneClean III Kit (GLASSMILK technology).
I find this is much better than Qiagen columns, better yield but it's much better at removing low yield contaminants.
hi LaiLab,
indead strange because we also use zymo-5 an zymo-25 columns fro PCR fragments, cDNA or genomic DNA.
yields around 80% are normal.
some questions to solve your problem,
- how do you know you have a 20% yield?
- if you do a etoh precipitation, what yield do you get then
- try and start with pure DNA follow the zymo manual, what yields do you have then?
- how do you determen the yield afterwards ? (gel electroforese or OD measurement).
good luck
So risking sounding silly, there are three point which I want to note,
The ratio of Q buffer volume to gel size must be correct. Too much gel, and the Q buffer will be too diluted to disrupt and dehydrate the water structure surround the DNA molecule. WItout proper disruption, the electrostatic attraction towards silicon within the column will not be strong enough to pull out the DNA. Making the silicon binding column as useful as a filter paper.
Adding isopropanol help. (It dehydrate the water structure)
and finally, the columns have a maximum binding capacity. Officially of 10 micrograms but my lab collegue has worked that to be around 15 micrograms. Anything more, and it'll just wash through with the solution.
Sorry to stray from the original posters query but in regards to Qiagens PCR purification kits, we also regularly use them here in our lab, BUT.............since we are targeting low copy number intracellular bacterial pathogens in whole blood we regularly elute our purified DNA in small volumes (around 30-40ul) still within manufacturers guidelines.
The problem;
Due to smaller elution volumes, residual ethanol from the supplied buffers is captured within the purified eluate and upon loading the agarose gel, we consistently see our samples "rise out" of their designated wells. Even using a 6x loading buffer at a ratio of 1:1, samples are still prone to leeching out of their wells. We have tried air drying the spin columns for 5 minutes prior to adding the elution buffer but still have the ethanol contamination problem.
Have you encountered this problem perneseblue, or anyone else??
The problem;
Due to smaller elution volumes, residual ethanol from the supplied buffers is captured within the purified eluate and upon loading the agarose gel, we consistently see our samples "rise out" of their designated wells. Even using a 6x loading buffer at a ratio of 1:1, samples are still prone to leeching out of their wells. We have tried air drying the spin columns for 5 minutes prior to adding the elution buffer but still have the ethanol contamination problem.
Have you encountered this problem perneseblue, or anyone else??
Yes, I have met this problem before a long long time ago. There are three things I would suggest.
1 - after PE ETOH wash, spin the tube for 1 min, then move the column to a new eppendorf and then spin for a futher 1 min. So there isn't even a trace of EtOH on the walls.
2- IF that fails... ethanol percipitate the DNA, wash it in 70%EtOH, dry the DNA in a hybridisation incubator at 65 Celsius for 3min or less. And resuspend in water. A hybridisation incubator has a fan which circulates the hot dry air, drying the sample faster.
3- IF DNA percipitation is not possible (your DNA sample is real realy low). Put eluted DNA (which has step 1 performer on it) into a hybridisation incubator. Leave for 3-5mins, the alcohol should have mostly evaporated.
And this is not really a method to reduce EtOH problem but a trip which prevents/reduces "The rising" horror in horrizontal gels.
When pouring you gel, wait until a skin forms on the surface, then lift the comb tp strech the skin and lower comb back down. Done correctly the skin should creep up the comb. When the gel is solidified, pull the comb out and a short collar will appear around the well. (Alternative is to fill the casting tray a little above normal, wait for gel to get viscous and then pipette excess gel out of tray. Collars also form around wells in this manner)
Next add the buffer, but load just enough buffer that the low collars of around the well are not submerged. They are clearly above the surface. Load sample, run at higher voltage to get the DNA into the gel. Once done, drop to normal runing voltage and add buffer to normal levels.
The ethanol contaminanted solution can't rise... there is no buffer above it to rise into. (due to the collars)
Hopes this helps.
Thanks Perneseblue.