Gel purification of vector DNA - low yield - (Feb/23/2009 )

Hi,

I seem to be getting a low yield (15ng/ul) when trying to purify my DNA from 1% agarose gels with the Qiagen PCR Purification kit. I have two vectors - in two seperate reactions 1 has been cut with AflII & the other with Sal I & ran on a gel. Got strong band for each cut vector on their respective gels but when i cut out & purify the yield seems to be really low. Any suggestions?

The yields from Qiagen gels are always low in our lab's experience. You could try a different kit - someone once suggested Zymo to me.

moorele on Feb 23 2009, 09:34 AM said:

I've never had a problem with the yield before. I usually get about 2ug/30ul so dont think its down to the kit. Plus others in my lab have been using it recently and havent had a problem with their yields.

I eluted in dH20 which a colleague has said usually results in a lower yield but not as low as i've gotten. I'm going to try eluting with Buffer EB diluted 1 in 5 with dH20 & see if that increases the yield.

might you have tried passing the QC buffer (yellow) which has the gel dissolve in through the column several times? I pass the solution 3 times. It gives the DNA more opportunity to bind to the column.

Also adding enough QC buffer to the gel is important. Too much is okay. Too little and the DNA doesn't bind to the silicon column as well.

Adding isopropanol, if you haven't already.

Have you tried eluting the DNA with EB buffer warmed to 68C?

perneseblue on Feb 23 2009, 05:15 PM said:

I've added isopropanol but havent tried passive the QC buffer through the column a few times! I'll give that a shot. I will also try warming the EB buffer! Thanks for those suggestions. I'll let you know if i have any success!

moorele on Feb 23 2009, 09:18 AM said:

Oh, another trick is to elute the column twice. Let say the EB volume you are using is 50ul. Break the volume of the EB buffer into two. Elute the column first with 20ul. Then a second time with 30ul. This does improve the amount of DNA eluted from the column.

perneseblue on Feb 24 2009, 01:33 AM said:

Or alternatively, u can elute in 50ul first, stand for 2 mins instead of the usual 1 min, and spin it down. then take the eluate and place it on the exact same spin column again and stand for 1 min and spin it down. u get better recovery this way.

Which kit do you use? If Qiagen, try adding the additional Na-acetate.

Or try invitrogen's kit, we got better yield with it.

Also, elution buffers need to have a pH of at least 8. ddH20 (surprisingly) is at pH of between 5 and 6.5...and in my experience, this has killed the yields of the column. The EB buffer from Qiagen is actually just Tris pH 8.0 (I think it's 10mM but not positive). If you are using water, try raising the pH to 8

I've been getting low yields too using the gel extraction kit from Qiagen.
I add 3M Na-acetate to the sample: didn't work.
I passed QG buffer twice: it washed away my DNA...

I don't know what to do...