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-   -   Purifying PCR product from 2% agarose gel (http://www.molecularstation.com/forum/protocols-methods-forum/19084-purifying-pcr-product-2%25-agarose-gel.html)

hilary cassidy 02-18-2007 01:46 PM

Purifying PCR product from 2% agarose gel
 
Hi

I've been trying to purify my PCR product from a 2% agarose gel all
week now as it needs to be purified prior to Topo cloning. I've used
both the QIAEX II Gel Purification kit by Qiagen and the Illustra DNA
and Gel Band Purification Kit by GE Healthcare. Each time I have
strong bands on the gel prior to cutting the bands, but everytime I
purify and run the DNA on a gel to check there is nothing!!!

Can anyone help?
Thanks
Hilary


Ednot 02-19-2007 01:03 PM

Purifying PCR product from 2% agarose gel
 
Ive had the same problem on a Min Elute GEl extraction Kit, this kit
is based on cartridges membrane, if you apply too much
sample or wrong pH, the sample will elute on the wash.

The QiaexII is based on charged silica, this item of the kit is very
labile
for my opinion, once i tried a new silica (newer lot) and it worked
just fine,
Once again check the pH of your sample.

A less partic method is to put your slice of gel with your band in a
eppendorff tube
and some TE buffer and let it a Max RPM on your centrifuge for 4
hours, remove the remaining agarose.
And precipitate the DNA with Ammonium Acetate 7M, pH 8.5 and then
Ethanol 100%.

Good Luck


On Feb 18, 10:46 am, "hilary cassidy" <[Only registered and activated users can see links. Click Here To Register...]> wrote:



chovek69 02-21-2007 08:34 AM

Purifying PCR product from 2% agarose gel
 
On Feb 18, 3:46 pm, "hilary cassidy" <[Only registered and activated users can see links. Click Here To Register...]> wrote:

Hi,
For purifying DNA from even 3% agarose gels I use a kit from Fermentas
based on NaI and silica. I've never had a problem with it and the
yield is quite amazing. There is a special isolation buffer from
agarose slices in TBE that according to the manual is harder.


StewJW 02-21-2007 04:58 PM

Purifying PCR product from 2% agarose gel
 
Invitrogen also have a gel extraction kit catalogue K2100-12, see
manual which has a trouble shooting guide which maybe helpful.

[Only registered and activated users can see links. Click Here To Register...]

They don't say what their gel solubilisation agent is.

Included is an interesting recovery table:

100 bp 1 μg 42.9 ± 10.7 %
400 bp 0.5 μg 95.4 ± 8.8 %
965 bp 0.5 μg 36.3 ± 5.8 %
5.4 Kb 1 μg 63.2 ± 12.6 %



chhoefer 02-22-2007 08:59 PM

Purifying PCR product from 2% agarose gel
 
On Feb 18, 2:46 pm, "hilary cassidy" <[Only registered and activated users can see links. Click Here To Register...]> wrote:

Hi Hilary, have you tried cloning without purifying. Usually TOPO
cloning does not require purification of the PCR product.
Rgds,
Christoph


ChenHA 02-23-2007 11:08 AM

Purifying PCR product from 2% agarose gel
 
Ednot wrote:


I stopped using QIAEX because it doesn't work well all the time,
especially for small fragments. I use Geneclean and it hasn't failed me
yet. Geneclean has a special kit for very small fragment (Mermaid if I
remember correctly), although trying this and the normal kits for ~200bp
fragments doesn't seem to make much difference (both worked equally well).

How big is your fragment? If you fragment is very small, check that you
haven't mistaken your oligos for PCR products.


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