efficiency of DNA cleanup columns - (Sep/12/2006 )
Hi guys,
I was wondering what brand of DNA clean up columns you use? I found QiaQuick spin columns seem to be of low yield. Although the procotol book claims that its recovery pecentage is around 80-90%, everytime I used the columns to recover the DNA fragment from gel, or cleanup from enzymatic reactions, I found the final yield is very low. I also tried the minielute columns, but seem to have the same problem.....
any recommendations or tips?
thanks,
VF
yeah, I think 90% is a pipe dream...but to increase the yield: heat the elution buffer to 60-70C, then let it sit on the column ~5min. this will vastly increase what you can pull off the column with qiaquick
thanks for the tips--I usually use Rnase-free water to elute DNA out---shall I heat that up as well?
as for sitting time, I usually do 3-min. and sometimes, I even try to elute twice (using the first eluted DNA solution to add onto column again, sit again and elute)--but even so, the yield is so low....
Today, to move my gene of interest into targe plasmid, I first setup the double RE digestion of TA-cloned plasmid carrying the gene, and target plasmid. Then I gel-purified the desired fragments and setup the ligation. Since the gel-purified fragments were of low concentration (via Qiagen column as well), I had to setup a big-scale ligation reaction (in 50ul volume, 200ng vector, 100 insert), then I cleaned DNA up with this Qiaquick column. When I spec the DNA with Nanodrop, almost no reading (and some samples for negative readings! how does that happen?). But I still managed to setup a kill degest (assuming my DNA was still present in the tube), transform the E.coli. I will see if any one or two colonies will pop up on the plates tomorrow--but I am not positive....
I just never could get a good yield using such columns,,,.
HI Vibrio fischeri,
I also think that 80-90% is a bit of a stretch. Just reading your blurb there are a couple of things that may help. First of all the recovery off the column seems to be proportional to the concentration of the starting material, faint band even poorer recovery. Also I too previously used water to elute my DNA, however, I found very poor recovery. I checked the pH of my water and found it to be very acidic, the columns require slighly alkaline solution for maximum recovery. I went back to eluting in Tris pH8 and improved my yield considerably.
Hope this helps
Scott
Scott is correct with the pH. The elution solution has to be neutral, leaning towards basic. Yields are bad with acidic (autoclaved) water. And I believe the Tris in the EB buffer also help with DNA stability.
Aside from Aimikins' suggestion of heating the elution buffer to 70 Celsius. I was wondering if you added isopropanol into the Q (orange coloured) buffer before running it through the column. That helps too.
And finally using the brute force and igonarance method, how about a really large scale PCR reaction. 4 X 50ul reaction. That should pass any hurdle.
For Qiagen kits, we also used to add Na-acetate (even if suggested to add if the buffer color changes).
Now we r using invitrogen's gel purification kit. This seems to give us better recovery than Qiagen kits.
thank you all for various tips--your points make sense to me--in the past I never measured the pH value of that RNase-free water always assuming it is around neutral.....
yes, I indeed add the isopropanol after the agarose slice is melted in the QC (yellow) buffer. But I did not add NaAc.
OK, I will take all your tips when using the Qiagen columns again...
yesterday, I ordered Zymo spin column--basically it is of similar or even same principle of the qiagen column. But the final elution volume is only 6-10ul and total treatment time is around 5 min. Another lab use it and says the recovery is good...so I plan to give a shot....
I will let you know if any colony shows up on the plates in yesterday's experiment....
thanks again...
VF
just an update... amazingly, I got a decent amount of colonies showing up in the antibioctic-containing plates. The negative where only water was added for transformation, no colony....
Those colonies look normal, healthy to me--I am now using the colony-PCR to screen...
I cannot understand why I still get colonies--since I got zero or even negative reading of ligation mix concentration via Nanodrop spec....(that was after the Qiagen Quickspin column cleanup).
anybody contributes any hints?--maybe the readings of Nanodrop is not sensitive to measure the very low amount of DNA....that is my guess....
thanks,
VF
Possibly,
you really only need 10pg of plasmid to get tons of colonies.
Alternatively it is time to send to send the old nanodrop for a good servicing and tune up.