Long-template PCR summary

Wendy S. Katz wkatz at POP.UKY.EDU
Wed Jun 18 09:26:42 EST 1997


Many thanks to all who responded.  A summary of the responses appears below
(including interesting tips about alternate enzyme sources and buffers).
=46WIW, I did side-by-side reactions in standard and thin-walled 0.5 ml
tubes; the standard tube gave a single band of the correct size with high
yield;  the thin-walled tube gave a moderate yield of a single band that
was way too small.  Aren't enzymes wonderful.  I used 20 sec denaturation
steps.

> Has anyone used the Boehringer Mannheim "Expand Long Template" kit with a
> thermal cycler other than the fancy Perkin Elmer job for which their
> protocols are optimized?  I have a Thermolyne Amplitron II and use
> thin-walled 0.5 ml tubes. My cycler transits between temperatures fairly
> rapidly, so I find the 10 sec. denaturation time in the Boehringer protoco=
l
> particularly troubling.   I'm interested to hear whether the protocol had
> to be modified extensively to work reliably in other machines (I'm going
> after a 10kb product).


=46rom: Dave Reiner

I don't know about long-range, but when I was in the Thomas lab, we
routinely used 10-15 sec denaturation times.  We used an MJR machine with
really fast ramp times, but used standard 0.5 mL tubes (thick walls).
Apparently this saves a lot of wear and tear on your Taq.  I don't think
ramp times matter that much.  Wall thickness is probably most important.


=46rom: Paul Baum

We've had great success with the kit using an MJ research PTC-200 cycler
which also has a fast ramp time.  We've also found that a manual "hot
start" (adding the polymerase mix to the reactions after they've warmed up
during the initial denaturation period) can sometimes increase success.

Also, we've found that a 1:16 volume ratio of Pwo:Taq works about as well
as the enzyme mix from the kit.  If you buy the Pwo from Boehringer and
the Taq from Promega, you'll save a lot over the kit price.


=46rom: French A. Lewis

        We have used the long PCR kit with an MJ thermocycler, and
lengthened the denaturation time to 30sec to ensure complete denaturation.


=46rom: Barth Grant

I have used the boehringer long PCR kit successfully on an old Ericomp
cycler.  I used 30 sec denaturing times.  I didn't try shorter ones.  I
don't think you you need to worry much until you get into the range of
>13kb.  I used extension times of 1.2 min per kb, since my machine also
lacks the "add time" feature.

=46rom: William R. Morgan

Have you also tried the BIOSCI's Methods & Reagents newsgroup?  You can
search their archives at http://www.bio.net/hypermail/METHDS-REAGNTS/ (and
you can also post a message to the group there as well, if you haven't done
so already).

=46rom: David M. Eisenmann

        I have used the BMB Expand PCR enzyme with MJ Research machines
extensively,
and have had great results.  I always use 94=B0 for 10 seconds as the first =
step.
I have found the annealing temp. to be the one that needs fiddling with (I u=
se
59=B0 30 seconds as a starting point).  I (as well as many others I Stuart's=
 lab)
have used it routinely in the 5kb - 20 kb range, and one guy in the lab
actually spanned a cosmid gap by long PCR. What's nice about it too is you c=
an
clean up the reaction and inject it directly into worms.  Good luck.

=46rom: Abby Dernburg

In agreement with Dave Eisenman, we have had success using the Expand Long
Template PCR kit with non-Perkin-Elmer machines:  in our lab we use
inexpensive Progene thermal cyclers with the same programs recommended by
Boehringer Mannheim.  But for long PCR I've also found that a different
reagent, the rTth XL enzyme and buffer from Perkin-Elmer Cetus, frequently
works better than Expand - either it gives a product where Expand fails or
it gives substantially higher yields.  It also has a proofreading activity,
so the products are suitable for cloning, injecting, etc.  Most of the
examples I've looked at are in the 6-7 kb range.  I think that the major
advantage is not the enzyme but rather the 3.3x proprietary buffer that's
supplied with it (it contains DMSO and glycine, but they don't tell you in
what proportions...).  You can order just the buffer at a small fraction of
the cost of the enzyme and check it out if you're interested.

=46rom:  Brian J. Libby

I saw your message regarding your long range PCR woes.  I also use the
BM kit and routinely amplify 12 kb fragments (so it can be done).  I use
the Progene cycler from Techne (about the cheapest available).  I don't
understand your point about trasmitting between temps rapidly.  I am
assuming that you mean slowly.  I really don't have any advice along those
lines as I follow the supplied protocol religiously.  However, i do have
some advice pertaining to other considerations (for what it's worth):

1.  thin-walled tubes is a must
2.  Vortex, vortex, vortex.  Vortex supplied buffer very well (one full
minute on highest speed).  Vortex final rxn mix for one full minute at highe=
st
speed.  You want to see foamy bubbles (trust me) at the end of this vortexin=
g.
3.  I add RNase to genomic template for an hour or so at 37C prior to additi=
on.
I'm not sure if this is essential, but I'm too superstitious to change.  I
guess that my thinking is that RNA binds free divalent cations, thus it's
beneficial to deplete RNA from sample.  Who knows?
4.  Use buffer 3 system and add at least 3 ul of 25mM MgCl2 to your MM2 in
addition to what is supplied in the buffer.
5.  Maintain a tight fit between rxn tube and cycler chamber (push down hard=
).
6.  This is probably highly variable, but it seems like an annealing temp
of about 8 degrees less than lowest primer melting temp is optimal.  Don't
be afraid to drop this too low.  I get product free of artifact bands at
annealing temps as low as 51.5C (I've never tried lower).








More information about the Celegans mailing list