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agarose gel purification of cloning vector

agarose gel purification of cloning vector - Protocols and Methods Forum

agarose gel purification of cloning vector - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 12-22-2005, 08:02 PM
mwcrepeau@hawaii.rr.com
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Default agarose gel purification of cloning vector



I've been trying to prepare a cloning vector for use in preparation of
genomic libraries. I digested pBluescript II with BamHI, incubated
with SAP and purified with the Qiagen QIAQuick kit. To test the vector
I produced a ~500bp fragment by digesting a PCR product with Sau3AI.
The digest was run on an agarose gel and the correct fragment was
excised and purified from the agarose using the QBioGene Gene Clean
kit. Because I find that the Gene Clean kit always renders DNA that is
unsuitable for UV absorption spectrophotometry (mysterious strong
absorption at ~240nm) I cleaned the insert DNA again with the QiaQuick
kit (after which I was able to quantify it reliably).

This vector/insert combination worked well (hundreds of white
colonies), but I also saw a lot of blue colonies that I thought were
probably due to uncut vector. I'd added a 10-fold excess of BamHI, but
I still could see what looked like a faint band of uncut, supercoiled
vector an a check-gel. So I prepared more digested vector, incubated
it with SAP, and then ran it on a gel and excised only the band
representing the linearized vector. Again I used the Gene Clean kit
followed by the QIAquick kit to purify the DNA from the agarose. I
expected the original high number of white colonies with a reduced
number of blue colonies, but what I got was almost no colonies of any
sort. Just one or two tiny blue ones. A repeat preparation of the
vector gave the same result.

It seems like somehow the gel extraction and purification process is
ruining my vector. And it seems to be specific to the vector side of
things, since an identical process was used to prepare the insert and
the insert works fine with non-gel-extracted vector. I suppose I can
learn to tolerate the high background of blue colonies, but the
above-described result has aroused my curiosity and I'd kind of like to
know what's going on. Before undertaking experiments to investigate, I
thought I'd solicit comments in this forum. Has anyone else had this
problem or does anyone have an idea what might be causing it?

All gels were prepared using 1X TAE and a garden variety agarose from
Invitrogen, with 0.025X Cambrex GelStar added for UV visualization.
Ligations were performed with T4 DNA ligase from Promega, overnight at
4C. Cells were commercially prepared DH5a, chemically competent at

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  #2  
Old 01-02-2006, 09:23 AM
newsnet customer
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Default agarose gel purification of cloning vector



Shouldn't you be measuring absorption at 260nm?
I heard that some commercial kits make quantitation via spectrophotometry
unreliable but I don't know why.


Examining your gel under UV light for too long when your trying to excise
your bands can ruin your vector.


Make sure your using well established protocols.

Cheers,
Sharp Tool



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  #3  
Old 01-02-2006, 09:54 AM
Christian Praetorius
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Default agarose gel purification of cloning vector

[Only registered users see links. ] wrote:


Why do you use 240nm instead of 260nm, which is usually used to
measure nucleic acids?


How long do you expose your gel to the UV light when you excise the
band? I learned to be as quick as posible, otherwise the UV light will
degrade the DNA.

Christian

--
Immerhin ist die relative Fluechtigkeit des Mediums Computer dem Gehalt vieler Bilder
in idealer Weise angemessen, waehrend man bei Papierbildern immer ueberlegt, ob "es das
Bild wert" sei. Oliver Corff, 23.08.05 in drf
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  #4  
Old 01-03-2006, 07:54 PM
mwcrepeau@hawaii.rr.com
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Default agarose gel purification of cloning vector

To clarify: the peak absorbance of the "mystery compound" is at ~240nm,
but it's a very strong absorbance so the shoulder of the peak is still
quite high at 260nm, therefor I don't trust the A260 measurement. The
instructions that come with the GeneClean kit mention that residual
glass milk can cause UV scattering and they don't recommend UV
absorption for quantitation. I don't know if that's what I'm seeing or
not, but the problem goes away if I treat the DNA with the QIAquick
kit. Then the spectrum looks entirely normal.

In cutting out the band I work as quickly as possible, but it still
probably takes 30 seconds. I've never timed it. I imagine
overexposure might reduce cloning efficiency, but even with extreme
overexposure isn't no colonies rather surprising? I am thinking I will
try another gel extraction kit and maybe a freeze/squeeze protocol to
see how they compare. Maybe I'll try staining with crystal violet.
Then I can cut the band from the gel without UV light.

Christian Praetorius wrote:

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  #5  
Old 01-03-2006, 08:51 PM
Tom Knight
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Default agarose gel purification of cloning vector

[Only registered users see links. ] writes:


The damage by UV is highly dependent on the wavelength. 254 nm light
is essentially instantly destructive. 305 nm light is questionable,
while 365 nm light is tolerable for some length of time. You might
want to look into the Clare "Dark Reader."

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  #6  
Old 06-17-2010, 09:33 AM
Pipette Filler
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Default Re: agarose gel purification of cloning vector

Peaks @ around 230 - 240 are indicative of huge salt amounds in your solution. Try to get rid of them by repeating you column purification or doing a phenol:chloroform OR just replace Qiagen kits!!
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