4 replies to this topic

[lucilius]

Dear all,

I am used to work with phenol-chloroform extractions, but recentely I tried a qiagen DNA extraction kit.
I am wondering if it is possible that the filters can get clogged up?
What is weird is that I see a big pellet in the recipient tube (the ones you trow away). So the celldebris does manage to go through the filter, but is it possible that the DNA is also "forced" through this filter with the cell debris? Or perhaps the cell debris is clogging up the filters?

I get very very low amounts of DNA.

thanks in advance

[jerryshelly1]

So you add your precipitating reagent (potassium acetate) to your sample, spin and transfer your aqueous solution to your column?  Are you very careful to not transfer over your precipitated SDS bound proteins?  This would of course interfere with the normal function of your Qiagen filter.  Slow down, do not filter the chunks with your aqueous solution.

[lucilius]

jerryshelly1, on 29 March 2013 - 11:10 AM, said:

So you add your precipitating reagent (potassium acetate) to your sample, spin and transfer your aqueous solution to your column?  Are you very careful to not transfer over your precipitated SDS bound proteins?  This would of course interfere with the normal function of your Qiagen filter.  Slow down, do not filter the chunks with your aqueous solution.

I use the qiagen kit protocol.
Not sure what you mean with adding precipitating reagent...
I lyse the cells, add the required buffers from the kit and then pipet this on the filter. The manual even says to pipet the pricipating stuff on the filter.

[phage434]

I think the confusion here is whether we are talking about spin minipreps or QTIP and Hi-Speed  column preps. For the QTIPs, after adding buffer P3, you should allow the precipitate to float to the top. This works best if the tube is chilled on ice. You can transfer the entire material to the filter, or (my preference) transfer only the clear liquid, avoiding most of the precipitate. This avoids the filter clogging. I don't think you lose much plasmid doing this (besides, it is a maxiprep, and you could have simply done a slightly larger one).

[lucilius]

phage434, on 30 March 2013 - 06:27 AM, said:

I think the confusion here is whether we are talking about spin minipreps or QTIP and Hi-Speed  column preps. For the QTIPs, after adding buffer P3, you should allow the precipitate to float to the top. This works best if the tube is chilled on ice. You can transfer the entire material to the filter, or (my preference) transfer only the clear liquid, avoiding most of the precipitate. This avoids the filter clogging. I don't think you lose much plasmid doing this (besides, it is a maxiprep, and you could have simply done a slightly larger one).

I was talking about the DNeasy Blood & Tissue Kit - Qiagen , perhaps I was wrong to call it filter tips.
The tips I speak of are the ones that catch the DNA.