2 replies to this topic

[siddharthsameer]

hello everyone
I have  a small doubt regarding the gel extraction purification. I cut of my fragment from the agarose and after weighing the weight of my fragments are 460mg and 550mg..but the Extraction kit says for the slice of more than 400mg we should use  more than one QIA column.I have never used in such conditions.You can anyone suggest me how to proceed with my extraction. I am unable to figure it out..
Quick reply would be really appreciated..
regards

[metionina]

You can use only one column for your fragment (460 and 550 is still ok), you can obtain a higher concentration of your purified DNA. Just load and spin your column more times.
Next times maybe try to cut smaller agarose slices.

[Trof]

Yes, it really useful to cut out all surplus agarose. Make cuts just above and below your band and don't leave sides. Also then cut out the bottom and upper part of the slice which doesn't shine under UV. I hardly get a slice weighting more than 100 mg.

The column is primarily limited by the amount of DNA, which is the same for small and big slice, but also by the amount of agarose that has to go throught the column. Maybe I would put there even more than 3x QG buffer to disolve the agarose even more than usual. You will need to centrifuge it repeatedly since there is only space for 800 ul in the column, maybe wash it with QG buffer after each spin, in addition to the final wash, so that the agarose will less likely clogg the column.