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Maddie's Photo

Maddie

Member Since 05 Nov 2009
Offline Last Active Jun 28 2012 08:07 AM

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#103235 What causes RNA/DNA to unbind from silica membrane?

Posted Maddie on 10 March 2011 - 08:38 AM

hianghao, on 07 March 2011 - 09:42 PM, said:

During nucleic acid extraction using column, the RNA/DNA bind to the membrane and in the last step, eluted by buffer/water? What is in the buffer/water OR what is not in the buffer/water that causes the DNA/RNA from being released from the membrane?

It has to do with salt and pH. For the DNA to bind to silica you need lots of salt and a low pH (below 7.5). To release the DNA you elute with a solution that is poor in salt and has a pH >7.5 (the EB in the Qiagen kit is at pH 8.0 I think).

#101688 PCR stopped working

Posted Maddie on 23 February 2011 - 09:27 AM

pedronog, on 23 February 2011 - 07:39 AM, said:

Hello everyone,

I'm going crazy with this...

I have a Taq from Biotools that comes with buffer with MgCl2, buffer without MgCl2 and MgCl2 solution.
I used to perform colony PCRs almost everyday and it always worked with the provided buffer with MgCl2. Always getting clean bands.
Untill one day when buffer with MgCl2 finished. I replaced this buffer with the buffer without MgCl2 and added MgCl2 separately so the reactions could have the same concentration of MgCl2 as the one provided already with MgCl2 (2mM).

The only thing that changed was the buffer and it just stopped working.
When I run the agarose gels, in the reactions from colonyPCR I get a huge amount of dna that does not go far from the well and the controls show a strong band with a shorter size than expected (~750bp). It seems that the expected band is there (~1.000-1.200bp) but it's too weak... Some smearing can also be seen... Negative controls (without dna) are 100% clean.

1 marker
2 negative control
3 control 1
4 control 2
5 control 3
6 control 4
7 control 5
8 colony PCR

what's happening???!

Thanks

I remember a company Tech once told me that it was important to vortex the magnesium quite a lot before the first use. I always vortex it for 7-15 seconds. I would try again. And if you want, you could also test out several MgCl2 concentrations (1.5 to 3mM).