Exo' III deletions for sequencing

[Norio Suzuki]

Clinton Grant says,
>I am interested to hear from people who have produced Exo' 
III deletions
>for sequencing: what are the best protocols

I get fine and peproducible result by  my protocol.
For ligaion step I can ligate within only 30 min with a good
efficiency by
Takara Syuzo's DNA ligation kit(1).

 I modifyed and mixed Exo/Mung Bean Nuclease Deletions 
protocol in 
Stratagene's pBLUESCRIPT DNA sequencing system and 
Red-book's protocol(2).

1. Digest your 10-20ug plasmid with 2 restriction enzymes 
(Kpn I and Sal I).
2. Extract with phenole/chloroform and ethanol 
precipitation.
3. Disolve plasmid in 30ul of 1xExo III buffer.
  4. Dilute 22ul of 10xMung Bean buffer in 170.5ul of water 
to final 192.5ul.
    Dispence each 17.5ul to 10 microtubes and "keep on ice".
    Transfer 2.5ul of #3 to above a microtube for time point
0 and place on ice
5. Incubate at temp.37C for 3 min.
6. Add 140 units of Exo III nuclease(about 0.5ul) and start 
timer immidiately.
   Continue to incubate at 37C.
7. Transfer 2.5ul of reaction mixture to #4 microtube at 
each 30 sec. and
   place on ice.
8. After that,heat #7 microtube to 68-70C for 15 min. to 
kill Exo III.
9. Add 6 units Mung Bean nuclease in 2ul of 1xMung Bean 
buffer.
10. Incubate at room temp. for 30 min.
11. Add 3.4ul of stopping buffer.
12. Make sure how may bases were cut by electrophoresis. 
    (2ul of reaction mixtur is enough to electrophoresis.)
13. Remaining reaction mixtures were extracted with 25ul of 
phenol/chloroform
    and precipitated by ethanol.
14. Dissolve deleted DNA in 10ul of Klenow mixture and 
incubate at 37C.
15. Add 1ul dNTP mix(each 0.25-0.5mM) and incubate at room 
temp. for 15 min.
16. Ligate 5ul of reaction mixture by Takara's ligation kit.
17. Transform E.coli. You get enough transformed colonies.

 10xExo III buffer
     500mM Tris-HCl,pH7.6
     10mM MgCl2
     10mM 2-mercaptoethanol
     
  10xMung Bean buffer
     300mM Na Acetate,pH5.0
     500mM NaCl
     10mM ZnCl2
     50%(V/V) glycerol
     
  stopping buffer
     1M Tris-HCl,pH8.0 15ul
     4M LiCl2 30ul
     20% SDS 6ul
     high viscosity
     
  Klenow mixture
     50mM Tris-HCl,pH7.5
     10mM MgCl2
     7units/ul klenow enzyme
     
(1) Hayasi,T., Nakazawa,M., Ishizaki,Y.,and Obayasi,A. 1985.
Influence of 
    cations on the activity of T4 DNA ligase in the presence
of polyethylene
    glycol. Nucleic Acids Res. 13:3261-3271
    
(2) Ausubel,F.e. et al (eds.) 1991. Current Protocols in 
Molecular Biology.
    Greene Publishing and Wiley-Interscience,New York

-----------------------------------------------------------
  Norio Suzuki
  Dep. of Molecular Biology,Tokai univ. School of Medicine
  E-mail norio at cc.u-tokai.ac.jp