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Ahrenhase

Member Since 03 Apr 2009
Offline Last Active May 13 2013 11:39 AM

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Topics I've Started

What's the chemistry behind flocculation when Qiagen P2 is mixed with N3

01 May 2013 - 10:12 AM

P2 (or the lysis buffer) is 200mM NaOH and 1% SDS. N3 (or neutralization buffer) is 4.2M GuHCl and 0.9M potassium acetate. What is floccing out? Is it the SDS which is bound to proteins?

Autoconversion of photoconvertable fluorophore

16 April 2013 - 11:59 AM

I'm working with the photoconvertable fluorophore, Kaede, and in neurons it seems to have a certain level of autoconversion. I don't think this is autofluorescence or an issue of overexposure because my controls don't autofluoresce red and I'm using the same exposure time as I do in other cell types (also transfected with Kaede), respectively.

Can you gene trap a particular gene locus

12 April 2013 - 03:17 PM

From what I'm reading, gene trapping sounds completely random. Can you target a gene of interest with this technique?

Can't find a recombinant after ligating

09 April 2013 - 12:07 PM

I'm trying to ligate a 1kb insert into a 5kb destination vector. Unfortunately, I am only able to use a single restriction site (Sal1); therefore, the cut plasmid can easily religate. I ligated insert and vector at a 3:1 ratio. I had to have screened over 60 colonies so far and not one was a recombinant (PCR and restriction digest analysis).

EDIT: And I am certain that my insert is being cut since I first put it into a topo construct.

Is ligation really necessary?

05 April 2013 - 04:39 PM

I came across a new method of cloning that involves generating 15-17 bp complementary overhangs on PCR products (http://www.biomedcen...1472-6750/11/92). In this method, the insert and the vector are PCR'd, treated with Dpn1 to remove the methylated template, and the pieces are directly transformed. The authors weren't sure of the method of exposing the stick ends, but suggested that the 3' exonuclease activity of high fidelity DNA polymerases expose these sites. If this worked well, then do we really need to ligate after a restriction digest?

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