lambda exo ssDNA sequencing
John H McDonald
mcdonald at ravel.udel.edu
Tue Aug 17 12:11:57 EST 1993
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 :-)
>Paediatric Molecular Genetics,
>Institute of Molecular Medicine,
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
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.
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