10 replies to this topic

[jamessmith01]

I am using using the Qiagen cleanup kits to purify DNA samples from ChIP (in the range of ng) for qPCR. However I'm not sure which of the following would be best for maximising the yield of a small amount of ChIP DNA:

1. Qiaquick and elute in 30 ul
2. MinElute and elute in 10 ul, then dilute to 30 ul

Both the Qiaquick and MinElute kits state the same predicted yields at ~80%. The protocols for the kits discuss the upper binding capacity of the columns, but not the loss at lower DNA amounts. I need more than the 10 ul elution volume of  the MinElute kit, as I will need to perform several qPCR reactions from each DNA sample.

Any ideas?

Edited by jamessmith01, 23 March 2010 - 03:40 AM.

[phage434]

Two elutions are much better for recovery than a single elution.  I'd recommend the minelute, eluted twice (or possibly three times, if you need the volume).

[jamessmith01]

Thanks for the info. Would you also recommend loading the sample twice?

[phage434]

I always put the lysate through twice.  It certainly can't hurt, and it is quick.

[Maddie]

Hi Phage

What do you mean by "putting the lysate through  twice"?

[jamessmith01]

Maddie, on Mar 24 2010, 02:16 PM, said:

Hi Phage

What do you mean by "putting the lysate through  twice"?

I meant, and I assume Phage meant, to load the sample on the column, spin, then load the eluate on the column and spin again.

[phage434]

Exactly.

[Maddie]

You mean elute twice with 15ul of EB each time, right?
That's what I would do.

Edited by Maddie, 26 March 2010 - 08:49 AM.

[jamessmith01]

Maddie, on Mar 26 2010, 05:48 PM, said:

You mean elute twice with 15ul of EB each time, right?
That's what I would do.

I didn't originally mean that, but based on the answers I will do at least two elutions.

[Maddie]

and don't forget to warm up the EB.

[Maddie]

BTW Phage, now I get it. Never thought about doing that. I'll try.
Thanks.