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Do you heat Tris HCl when you elute from silica?


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3 replies to this topic

#1 Maddie

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Posted 20 January 2011 - 07:52 AM

You know sometimes you do things for years and suddenly you wonder why? Where did this come from?
Well, it's happening to me. I've been using the Qiaquick and MinElute kits from Qiagen for years and usually heat they EB buffer (which is Tric HCl) when I elute. Then recently I came across protocols where they didn't heat and wondered if heating was wise. I can't remember where I got the idea from. I've probably seen this in a kit (I think the MiniBlood kit uses hot AE to elute), but when I read the manual from the MinElute, they don't advise to heat. Only to repeat the elution a second time if we want to increase the DNA yiel a bit.
Now I wonder if using EB at 70 degrees will hurt, help or do nothing at all. What do you think?

Question number two, also related to elution and silica. The most recent protocols of my field advise to add 0.05% Tween to the EB buffer when eluting your DNA from the silica. Adding a detergent to my extract doesn't sounds like a good idea to me, especially since I'll use enzymes in the next steps, but the guys who published that are really good. Any idea?
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#2 phage434

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Posted 20 January 2011 - 11:10 AM

If you look at the buffers for restriction enzymes and other enzymes by suppliers, many of them already have mild non-ionic detergents such as tween. Heating the elution buffer assists in elution. I think the reason it is not widely suggested is that it is one more step in what suppliers are hoping to make an easy process.

#3 hobglobin

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Posted 20 January 2011 - 11:17 AM

In Qiagen's Blood and Tissue Kit it's mentioned in the Troubleshooting section for some of the protocols to increase elution efficiency.
I regularly did it in such kits with EB/AE buffers, because DNA amounts are always low (tiny insects).

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#4 Maddie

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Posted 20 January 2011 - 12:44 PM

Thank you very much gentlemen.
It's a bit weird that they mention heating in the extraction kit's troubleshooting chapter and not in the MinElute one.
Not very logic if you ask me :rolleyes: .
Anywayyy, I'll take your word and go on heating my buffer AND I'll keep the Tween. THANK YOU.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein




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