lambda exo ssDNA sequencing

[John H McDonald]

In article <1993Aug17.121221.1 at molbiol.ox.ac.uk> awalley at molbiol.ox.ac.uk writes:
>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 hwere unablle to get a free sample from Pharmac :-)
>
>Andrew Walley,
>Paediatric Molecular Genetics,
>Institute of Molecular Medicine,
>Oxford, U.K.


This procedure is described in Higuchi and Ochman, Nucleic Acids Research
17:5865.  It works very well, and it's easy enough that you should be
embarrassed to use a kit.  Lambda-exonuclease is available from GibcoBRL
or Pharmacia.  Here's the protocol:

1. Do a 100 ul PCR reaction using one phosphorylated primer and one
regular primer.  
2. Add 0.5 units lambda-exonuclease plus 10 ul lambda-exo supplement (775 mM
glycine, 278 mM KOH, 5.8 mM
MgCl2).  This supplement plus the PCR buffer makes lambda-exo buffer.
Making a cocktail of enzyme plus supplement doesn't hurt the enzyme, at
least not for a few minutes.  Incubate 30 minutes at 37 C.
3.  Do one phenol/chloroform extraction, then one chloroform extraction.
4.  Precipitate by adding 50 ul 7.5 M NH4OAc, 150 ul ethanol.  Five
minutes at room temperature, then spin 15 minutes.  This is a selective
precipitation; these conditions will precipitate large DNA, but not
primers or unincorporated nucleotides.
5.  Wash with 1 ml 70% ethanol, dry, and resuspend in T.E.  A typical PCR
reaction will yield enough DNA for 2 or 3 sequencing reactions.