PCR and Agarase

davis at neb.com davis at neb.com
Tue Mar 9 13:43:26 EST 1993


>Dear Netters,
>
>This is to confirm that DNA gel-purified with beta-agarase can't be
>amplified by PCR.  I did a control experiment with a template and
>primers that ALWAYS work in which I loaded the template onto a LMP gel
>and purified it with a beta-agarase digestion.  There was no
>amplification.
>
>I have just spent a month trying to amplify my ligation product. 
>Because of a sin of omission in the agarase instructions the
>amplification was doomed not to work :-(.  The instructions say
>
>   Beta-agarase I acts by cleaving carbohydrate bonds, freeing trapped
>   DNA, and producing carbohydrate molecules which can no longer gel. 
>   The remaining carbohydrate molecules and Beta-Agarase I will not, in
>   general, interfere with subsequent DNA manipulation including:
>   restriction endonuclease digestion, ligation and transformation.
>
>And, of course, excluding PCR.
>
>Someone has written to me telling me his lab has found exactly the same
>thing with the american version of the enzyme called gelase.  I'm rather
>miffed that I wasted so much time for nothing.
>
>Others, beware!
>
>/--------------------------------------------------------------------------\
>< Ramin Charles Nakisa, "She walks in beauty Tel:    071 589-5111 x 6729   >
>> Biophysics Section,   As the night of      FAX:    071 589-0191          <
>< The Blackett Lab,     starry skies and     EMAIL:                        >
>> Imperial College,     cloudless climes..."         ramin at ic.ac.uk        <
>< London SW7 2BZ.                                    mbrcn at seqnet.dl.ac.uk >
>\--------------------------------------------------------------------------/


First, let me explain that as a member of this newsgroup, I do not
'represent' my employer, and that my opinions do not necessarily reflect
those of my employer or anyone else for that matter.

As to the above quote (written by me, extracted from a product data card by
Dr. Nakisa), the 'sin of omission' was not in the writing, but in the
doing.  Experiments testing the use of agarase purified fragments in PCR
reactions have never been done here.  We do have some anecdotal data
(observations, not systematic studies) which may help to explain why PCR of
agarase purified fragments is problematic.

1)  Agarase purified M13 ssDNA can only be sequenced using Klenow after
phenol/chloroform extraction.

2)  Cycle sequencing with Vent exo- DNA polymerase gives nice results with
agarase purified fragments.

My guess is that polymerases are differentially susceptible to either
agarase itself or to the small amount of undigested agarose which may
remain. 

My apologies if you found this misleading, believe me, it was not
intentionally so.  I might add that most reputable manufacturers will be
happy to provide any additional technical support they can if you are
having trouble.  Call them (us) first.

ted    
---------------------------------
Ted Davis
New England Biolabs
davis at neb.com




More information about the Methods mailing list