Exonuclease VII vs Mung Bean Nuclease

Jorg Kirberg Kirberg at nki.nl
Thu Aug 6 13:08:00 EST 1998


In article <35C9C2D0.2723 at chugaibio.com>, dspinella at chugaibio.com,
@chugaibio.com wrote:

> Please don't misunderstand my earlier postings -- I did not mean to
> imply that MBN will not work at all for polishing single-stranded
> overhangs.  It surely does.  However as you note, one needs to control
> the reaction carefully (for example, by making serial dilutions of MBN
> as you have done).  At best, this entails a bit more work than using
> Klenow or T4 Pol to do the same job. Moreover, if 25% of your clones
> were derived from incomplete MBN digestions in which the overhangs were
> repaired by the bugs, you can bet that there were many more such
> reaction products that failed to clone altogether. This also suggests
> that your "optimal" MBN concentration was pretty low, so its not
> surprising that you didn't see any "chewback" reaction products.

No misunderstanding at all, just to say that it might work better than
anticipated. BTW, you could also put the argument the other way
around: If you get religation products with a 2bp 'sticky' overhang
in only 25 % of the cases, and these should be be relatively more efficient
compared to the blunt-end ligation, it indicates a preponderance
of the desired blunted DNA.
> 
> [...] This "gut feeling" is derived not only
> from conventional wisdom as dispensed in the popular cloning manuals and
> literature, but from experience with producing cDNA libraries by adding
> adapters to MBN or T4 polished cDNA. I have always obtained many more
> clones after switching to T4 Pol (from MBN) to polish the cDNA although
> I have never compared the polishing techniques directly with the same
> constructs. Has anyone out there ever made such a head-to-head
> comparison? Do let us know the results. Cheers. --Dom Spinella

That would be a fair comparison. But admittedly I assume it will not
turn out to be in favour of MBN ...

jorg


E_mail kirberg at nki.nl



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