Summary-lamda exo ssDNA prep

awalley at molbiol.ox.ac.uk awalley at molbiol.ox.ac.uk
Mon Sep 20 10:12:33 EST 1993


Hi,
   For those of you who replied to my question about the
Pharmacia ssDNA prep kit thank you and there follows an edited
version of the responses I received, together with my comments
on using the kit so far. If you have any tips on using the kit
please note that there a couple of people mentioned below who
would also like to hear from you, as well as myself.

Andrew Walley,
IMM, Oxford, England
--------------------------------------------------------

From:     MX%"dnaseq at neb.com" 18-AUG-1993 15:43:12.64

Organization: new england biolabs

In article <1993Aug17.121221.1 at molbiol.ox.ac.uk>, you wrote:
> 
> Pharmacia PCR Template Prep:
> 
> As we are having problems with direct sequencing of PCR
products we are looking for ways to improve the template. This
kit, advertised in Nature Genetics recently, offers the option
of ssDNA sequencing using a dsDNA PCR product as the starting
material. This is achieved by selectively degrading one strand
using lambda exonuclease.
     Does anyone have any opinions on this product or experience
of it in practice as we were unable to get a free sample from
Pharmacia :-)
> 
> Andrew Walley,
> Paediatric Molecular Genetics,
> Institute of Molecular Medicine,
> Oxford, U.K.
]
reply:  i know you do not want to be bombarded with company
litany, but the Circumvent sequencing kit works really well on
PCR products, provided they are cleaned up appropriately.  feel
free to contact me if you have further interest.
regards,  barton slatko, head of DNA sequencing group at neb
-----------------------------------------------------------------
From:     MX%"GBGA13 at vms3.gla.ac.uk" 17-AUG-1993 17:19:49.63

I was sucker enough to buy this kit as it's on a free offer,
coupled to one of their sequencing kits...sorry, not free,
special, i.e. 100 quid less than both together.  You don't get
much.
     More to the point the minicolumns and sephacryl provided
are, to judge from the manual, capable of only purifying a small
amount of product. So, for SSU rRNA gene sequencing, which we are
doing, with ca. 8 internal primers, a lot of sephacryl would have
to be used. It does not look like a good buy, since the method
is basically well-known from the literature.
Oh yes, the kit doesn't even include the T4kinase!!

We tried the method once, based on the literature.  The
exonuclease product looked OK on gels, but wouldn't sequence, so
we went straight back to our usual Centricon & a/s amp. method.

I'd be interested to see any summary that you post.
Bernie Cohen
Genetics
Glasgow

Bernie, I should have listened :-)  The columns do in fact have
a low capacity for purifiying your DNA, so much so that an easily
visible band on a gel became invisible by the end of the
procedure. We are now using glass milk to purify the DNA.
-----------------------------------------------------------------
From:     MX%"jgraham at bronze.ucs.indiana.edu" 17-AUG-1993 

You should have a look at USB's newletter "Comments" vol. 18 no.1
(summer 91) for details on the use of T7 phage Gene6 Exonuclease
for this pupose. Call 1-800-321-9322 and ask for a copy. The
article contains a nice set of references and focuses on the
direct sequencing of Lambda.

Good luck,

Jim
J. E. Graham
Biology and Chemistry Departments
Indiana University Bloomington
-----------------------------------------------------------------
From:     MX%"kenward at molbiol.ox.ac.uk" 17-AUG-1993 13:11:59.85

I don't know about the kit you mention, but have you contacted
Martin Richards in the Collagen genetics group - he uses direct
PCR sequencing all the time.
     Cheers,
               Nigel Kenward
-----------------------------------------------------------------
From:     MX%"maiorano at molbiol.ox.ac.uk" 17-AUG-1993 12:34:51.42

Hi,

I have never used the Pharmacia kit but I have good experience
to do direct sequencing of dsPCR products by asymmetric PCR. This
method worked fine in my hands and produce good quantities of
ssDNA to be sequenced. I can send you the detailed protocol and 
the reference of the method if you wish.

Good luck

Domenico Maiorano
Department of Zoology
Oxford University
England

We had little success with asymmetric PCR which was why we were
looking at the Pharmacia kit.
-----------------------------------------------------------------
From:     MX%"n.salazar at lshtm.ac.uk" 18-AUG-1993 10:41:03.16

Dear Andrew,
                I am also very interested in the comments about
the use of the PCR pharmacia kit to sequence PCR products. You
mention that you have problems with the template for sequencing,
I hope I will not have this problem, I just started getting good
quantity of my PCR product to do the lambda Exo digestion. Could
you please send me the comments that you are receiving about it
for me to be aware of possible trobles that I might find ?? This
is the first time that I am doing this protocol.

Thanks very much and I look forward to hearing from you.

                        N. Salazar
**************************************************************
Nelson Salazar                      *  E-mail:
n.salazar at lshtm.ac.uk
Dept. of Medical Parasitology       *    or   
nsalazar at mrc-crc.ac.uk
Lon. Sch. Hyg. & Trop. Med.         *  Phone : 071 927 2468
(Office)



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