Gel purification of vector DNA - low yield - (Feb/23/2009 )
Kami23 on Mar 12 2009, 09:22 AM said:
tuhin_genes on Mar 11 2009, 02:19 AM said:
moorele on Feb 23 2009, 09:34 AM said:
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?
hello
when you are gel isolating the bands, for how long you were exposing the vector DNA on the gel doc? It might so happen UV nicks the DNA when exposed so long , moreover you should try the preparative UV which has got longer wavelength than the shorter ones.
It may also happen that the Ultra salt may be too old ( NaI) to dissolve the gel completely and the silica beads may be old to bind to the DNA. try to vortex the silica mixture with the DNA so that the Brownian motion continues and when you precipitate by centrifuging , you get a lots of silica attached to the DNA.
cheers
Hey,
I have always hated the column extraction kit from Quiagen because the yeild is always so low! but they do a kit which uses a resin called QiaxII which i prefer and has always been ok for me... its a bit more fiddly but well worth it!
the one from viogene also depresses me. sometimes i use pcr kit to purify digested vector, no band prep at all, the yield is much better.
I can also confirm that both the Qiagen gel extraction kit as well as its gel purification kit fall well short of their recovery claims of 90-95%. To give you some numbers:
(*40µg is well above their stated maximum binding capacity per column of 10µg.)
<*>40µg* in 1µg out = 2.5% 
<*>10µg in 0.5µg out = 5% 
I tested 2 batches of Qiagen kit, NaAc, the 1min wait during elution, eluted with EB not to get the bad recovery due to pH. People in my lab tell me though that it worked better before. They also tried the warming and other tricks to no avail.
My yields have increased since I started using much more solubilising solution than recommended .
swanny on Oct 28 2009, 09:04 AM said:
we hv started using a kit from another company called RBC. works really well
moorele on Feb 23 2009, 09:34 AM said:
when you are gel isolating the bands, for how long you were exposing the vector DNA on the gel doc? It might so happen UV nicks the DNA when exposed so long
Hi
How long should DNA be exposed to UV? is 5 min so long?
rukiye on Feb 9 2010, 05:55 PM said:
moorele on Feb 23 2009, 09:34 AM said:
when you are gel isolating the bands, for how long you were exposing the vector DNA on the gel doc? It might so happen UV nicks the DNA when exposed so long
Hi
How long should DNA be exposed to UV? is 5 min so long?
I would say it's too long. I try not to go above 30 seconds and even that is pushing it. I've never heard a specific time, but just to go as fast as possible.
ruiN on Mar 7 2009, 09:52 AM said:
Why would that help?