Gel purification of DNA bands-any alternates? - (May/29/2007 )
I typically load 13 ul in my agarose gel wells. Then clean the bands with mobio kit. The problem is the the kit allows to place only 0.2gm of gel band on its spin filter to melt it. So if its more than 0.2gms, I have to cut the gel band and load it on a 2nd column and after more cleaning and spining steps, elute it with a buffer. In the end I get very less DNA (like 7-9 ng/ul).
I need two bands, 3kb from one sample and 1 kb from another sample for a subsequent Roche ligation step.
I am wondering if there is any other way of purifying the DNA from the gel to use it in ligation.
Somone suggested that I simply melt the gel, take 1 ul from it and dilute it with water and use it in my subsequent ligation.
i would like to suggest you to change your kit for purification of DNA from agarose gel so that you could get high yield.
If you just need a little, as in cloning, you could cut out your band, place the gel in the top of a filtered 100 ul tip, cut the bottom off the tip so it can fit into a 1.5 ml microcentrifuge tube, put the tip into the tube, and centrifuge at 13K rpm for 30 seconds or more, and then use the liquid that passes through the filter for your ligation. Fast and cheap. Works for me.
why not run both pieces of agarose through the same column? As long as the column's DNA binding capacity is not reached, you can continue passing melted agarose through the filter.
cuation : if agarose is not properly dissolved, you'll block your column.
Using an electroelution may give you the possibility to purify greater quantities.
I use up to 400mg in one single column, but heat little extra and spin immediately after loading.
using agarase may you help you to degrade agarose.
An other possibility is to prefilter your melted preparation in smaal piece of glass wool