Exo' III deletions for sequencing
norio at tokaigw.cc.u-tokai.ac.jp
Sat Feb 1 02:14:48 EST 1992
Clinton Grant says,
>I am interested to hear from people who have produced Exo'
>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
Takara Syuzo's DNA ligation kit(1).
I modifyed and mixed Exo/Mung Bean Nuclease Deletions
Stratagene's pBLUESCRIPT DNA sequencing system and
1. Digest your 10-20ug plasmid with 2 restriction enzymes
(Kpn I and Sal I).
2. Extract with phenole/chloroform and ethanol
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
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
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
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
10xMung Bean buffer
300mM Na Acetate,pH5.0
1M Tris-HCl,pH8.0 15ul
4M LiCl2 30ul
20% SDS 6ul
7units/ul klenow enzyme
(1) Hayasi,T., Nakazawa,M., Ishizaki,Y.,and Obayasi,A. 1985.
cations on the activity of T4 DNA ligase in the presence
glycol. Nucleic Acids Res. 13:3261-3271
(2) Ausubel,F.e. et al (eds.) 1991. Current Protocols in
Greene Publishing and Wiley-Interscience,New York
Dep. of Molecular Biology,Tokai univ. School of Medicine
E-mail norio at cc.u-tokai.ac.jp
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