How to improve yield in DNA-purification from agars electrophoresis? - (Mar/29/2014 )
As title states, how can I improve the yield?
I go from 300ng/ul down to barely 20ng/ul.
I tried to incubate longer time at RT (too evaporate the ethanol from the washing step, but at the same time not too long to risk getting over-dried). Also tried to centrifuge at higher speed and for 2min. And to divide my elution down to 2steps (first 20ul and then 10ul).
Not much improvement so far.
I am using Ge Healthcare Illustra Agaros Purification Kit.
There isn't a whole lot that you can do - yields of 10% or less are very common with gel extractions. The reason is that agarose is a sugar based molecule similar to DNA, so separating the two is quite hard.
You can do a few things
1 - increase the amount of capture buffer you are using. Increase by 50%.
2 - you can pass the capture buffer through the column two or three times.
3 - you can add isopropanol to the (capture buffer + agarose) solution (100ul for every 300ul)
4 - you can use warm elution buffer.
5 - you can do two elutions (which you have already done).
but that said, you do take hit from using a column.
Also have a look on older threads, this is a common problem:
and so on...