Membrane of Qiaquick remain coloured - Problem of elution - (Feb/05/2007 )

Dear All,

Has anyone ever met a problem in the elution with Qiaquick PCR
purification kit ?.
Traditionally, for a direct labelling without amplification, we
hydrolysed with 1M of NaOH, neutralised with 1M Tris-HCL PH:7.5, and
purified with Qiaquick without problem. But recently, We notice that
the membrane of the Quiaquick remain coloured and the elution isn't
very efficient.
We have changed the kit of Qiagen, the buffer, NaOH, Tris ,... but the
membrane of the quiaquick remain coloured.
When we purified with an another system, microcon YM-30, we noticed
that the probe seems to be precipitate, this explains maybe that we
can't elute the probe with the Qiaquick...
All suggestions are welcoming.
Best regards

Hi

What do you mean when you say that membrane is coloured? New Quiagen kit comes with the binding buffer that will show you the efficiency of neutralisation. It has pH indicator that will go blue if your sample is basic and red if yoyr sample is acidic. If after you add buffer your sample is not yellow, you should normalise your 1M acid you use to neutralise NaOH, to 1M base (NaOH). If their concentrations are normalised so that eg. 1ml of 1MHCl and 1ml of NaOH give you neutral pH you shoud not have any colour after elution. Do ethanol wash step as fast as possible as ethanol is not good for cy labeled sample.

Lots of luck