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lucilius

Member Since 15 Apr 2009
Offline Last Active Today, 04:04 AM

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microbiology, biotechnology

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Posts I've Made

In Topic: Role of sodium acetate in DNA extraction/precipitation

Today, 01:21 AM

bob1, on 23 May 2013 - 12:50 AM, said:

Most DNA extractions have a chaotropic step which denatures proteins which are then removed before the DNA precipitation step.

Ok I see what you mean.
When you do a genomic DNA extraction/precipitation you have an extra step to remove the proteins before you precipitate the DNA.

In Topic: Role of sodium acetate in DNA extraction/precipitation

Yesterday, 11:15 PM

bob1, on 22 May 2013 - 01:02 PM, said:

The role is to increase the number of ions in solution to a point where the DNA can be precipitated by the addition of an alcohol primarily. In some extractions such as plasmid preps, it is used to neutralize the alkaline component of the lysis (step 2 NaOH and SDS) and precipitate the proteins and genomic DNA from this step, again through ionic strength.

Yeah, I understand this.
But in the case of genomic DNA: if you precipitate the DNA with the proteins, does this cause problems? Or is it not a problem to have a lot of proteins too for (for example) a DNA sequencing test?

In Topic: Ambigious about the acceptance

Yesterday, 09:09 AM

Mad researcher, on 09 April 2013 - 10:30 AM, said:

Thanks

keeping my fingers crossed...

Did you get it?

In Topic: clogging up qiagen filters

30 March 2013 - 06:35 AM

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.

In Topic: clogging up qiagen filters

30 March 2013 - 02:10 AM

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.

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