How to improve yield in DNA-purification from agars electrophoresis? (View forum version)



Biologystudent

Posted 29 March 2014 - 12:07 PM

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.

 


bob1

Posted 29 March 2014 - 03:04 PM

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.


perneseblue

Posted 30 March 2014 - 06:56 AM

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.


hobglobin

Posted 30 March 2014 - 09:12 AM

Also have a look on older threads, this is a common problem:

http://www.protocol-online.org/forums/topic/28767-low-yield-pcr-product-after-gel-purification/?hl=%2Bgel+%2Bextraction#entry152204

http://www.protocol-online.org/forums/topic/30866-dna-extraction-from-the-gel-low-yield/?hl=%2Bgel+%2Bextraction

http://www.protocol-online.org/forums/topic/29890-no-dna-after-gel-extraction/?hl=%2Bgel+%2Bextraction

 

and so on...