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small fragment of DNA PCR Product elution kit difficulty

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Old 09-16-2006, 03:41 AM
V Arunachalam
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Default small fragment of DNA PCR Product elution kit difficulty



Hi,
I am facing problem while elutung a PCR product amplified from genomic DNA of 200 bp size fragment. The DNA recovery is low. Could anyone suggest me good kit / modification in the protocol for getting high recovery of DNA.



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Old 09-16-2006, 06:12 PM
Jose de las Heras
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Default small fragment of DNA PCR Product elution kit difficulty


"V Arunachalam" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ] .net...


Try digestion of the agarose by enzymes such as Agarase/AgarAce etc
(different names for different companies). That always worked very well for
me.

A simple electroelution may work too (just put the cut band into a small
piece of dialysis tubing with a little buffer, and run it for a few minutes
in teh tank... recover the DNA in the buffr by precipitation).

Lately I have been needing to purify and clone many PCR fragments, and I got
tired of the columns and their poor yield. I simply freeze the bands for
15-20 minutes, then thaw them and spin them in an eppendorf at max speed for
a couple of minutes. Use the "juice" for ligations, it works very well.

I hope any of these help.

Jose
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Old 09-16-2006, 11:21 PM
DK
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Default small fragment of DNA PCR Product elution kit difficulty

In article <[Only registered users see links. ].net >, V Arunachalam <[Only registered users see links. ]> wrote:

What kit/protocol do you use? I'd think it's more likely that it is binding
step, not DNA elution, that is inefficient for such relatively small fragment.
In which case increase binding bu using higher gel to chaotropic salt
ratio. As an example, with Qiagene's gel extraction spin kit, use 5V QG
solution instead of recommended 3V (and do use isopropanol as
suggested).

DK
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Old 09-16-2006, 11:58 PM
ChenHA
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Default small fragment of DNA PCR Product elution kit difficulty

On Fri, 15 Sep 2006 20:41:11 -0700 (PDT), V Arunachalam
<[Only registered users see links. ]> wrote:


Try Geneclean (from Q-Biogene). Always found it reliable, and it
gives reasonable yield, unlike Qiagen which I found to be unreliable
(sometimes good, but sometimes shitty yield especially for small
fragments). Geneclean also has the Mermaid kit which is specifically
designed for very small fragments, but this is not necessary for
200bp. We recently tried purifying a 150 bp PCR product and got
reasonable good yield from both the Geneclean Turbo and Mermaid kits.
You can purify directly from the PCR reaction or from agarose gel.



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