遺伝子材料開発室 - DNA BANK - > リソースの取り扱い > 微生物ゲノムDNAの調製

微生物ゲノムDNAの調製

Preparation of Genomic DNA

  1. Suspend bacteria cells in 8 ml of [0.7 M NaCl, 0.1 M EDTA (pH 8.0)] in 50 ml conical tube.
  2. Add 1 ml of 10 mg/ml lysozyme in PBS.
  3. Add 30 ul of 100 mg/ml labiase.
  4. Incubate mixture in 37oC water bath for 1 hour.
  5. Add 1 ml of [0.5 M tris-Cl (pH 7.5), 5% SDS].
  6. Add 25 ul of 10 mg/ml protease K.
  7. Incubate mixture at 60oC for 1 hour.
  8. Extraction of DNA with phenol.
  9. Extraction of DNA with phenol-chloroform.
  10. Extraction of DNA with chloroform-isoamyl alcohol (24:1).
  11. Precipitation of DNA with cold ethanol.
  12. Wash precipitated DNA with 70% ethanol.
  13. Dissolve DNA in 600 ul TE buffer with 1ul of 10 mg/ml RNaseA.
  14. Incubate DNA at 37oC for 30 min.
  15. Add 5 ul of 10 mg/ml protease K.
  16. Incubate DNA at 60oC for 1 hour.
  17. Extraction of DNA with phenol.
  18. Extraction of DNA with phenol-chloroform.
  19. Extraction of DNA with chloroform-isoamyl alcohol (24:1).
  20. Add 100 ul of 3 M sodium acetate (pH5.5).
  21. Add 1.2 ml cold ethanol.
  22. Wash precipitated DNA with 70% ethanol.
  23. Redissolve DNA in 200 ul TE buffer.
  24. Dilute 2 ul of DNA into 400 ul dH2O and analyze OD260.

Reconstitution of DNA

  • In the case you received plastic vials contains precipitated DNA with ethanol.
  • A precipitant might stick to the lid or wall during transportation.
  • Before opening the tube, please spin down a minute or two at 5,000 g (~7,000 rpm, table top microfuge).
  • Please remove ethanol with pipet (DO NOT tooch precipitate!)
  • Immediately after removal of ethanol resuspend the DNA in an appropriate amount of TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA), and dissolve the DNA pellet by gently tapping with fingers (DO NOT shake vigorously! A little amount of ethanol won’t affect to many applications).
  • If the DNA is difficult to solve, leave the vial at 4 degreeC overnight to allow the DNA to dissolve.
  • Once reconstituted in TE buffer, the DNA should be stored at -30 degreeC.

Related Information

Primers for PCR of rRNA gene

Bacteria 16S rRNA
Name of primer Sequence
EB-20F 5′ AGTTTGATCCTGGCTC 3′
EB-350F 5′ TACGGGAGGCAGCAG 3′
EB-1100R 5′ AGGGTTGCGCTCGTTG 3′
EB-1400R 5′ ACGGGCGGTGTGTAC 3′
EB-1530R 5′ AAGGAGGTGATCCAGCC 3′
Reference of primer
  • Itoh, T., Kudo, T., Oyaizu, H. and Seino, A.
    Two new species in the genus Actinomadura: A. glomerata sp. nov., and A. longicatena sp. nov.
    Actinomycetologica 9: 164-177, 1995.
    DOI: 10.3209/saj.9_164.
  • Namwong, S., Tanasupawat, S., Smitinont, T., Visessanguan, W., Kudo, T. and Itoh, T.
    Isolation of Lentibacillus salicampi strains and Lentibacillus juripiscarius sp. nov. from fish sauce in Thailand.
    Int. J. Syst. Evol. Microbiol. 55: 315-320, 2005.
    PMID: 15653893. DOI: 10.1099/ijs.0.63272-0.
Archaea 16S rRNA
Name of primer Sequence
A-20F 5′ TCCGGTTGATCCTGCCG 3′
A-340F 5′ CCCAGGCCCTACGGG 3′
A-520R 5′ GTATTACCGCGGCGGCTG 3′
A-1100R 5′ CGGGTCTCGCTCGTT 3′
A-1400R 5′ GACGGGCGGTGTGTGC 3′
Reference of primer
  • Itoh, T., Suzuki, K. and Nakase, T.
    Occurrence of introns in the 16S rRNA genes of members of the genus Thermoproteus.
    Arch. Microbiol. 170: 155-161, 1998.
    PMID: 9683654. DOI: 10.1007/s002030050628.
  • Itoh, T., Suzuki, K., Sanchez, P. C. and Nakase, T.
    Caldisphaera lagunensis gen. nov., sp. nov., a novel thermoacidophilic crenarchaeote isolated from a hot spring at Mt Maquiling, Philippines.
    Int. J. Syst. Evol. Microbiol. 53: 1149-1154, 2003.
    PMID: 12892143. DOI: 10.1099/ijs.0.02580-0.
Fungi 26S rRNA
Name of primer Sequence
ITS5 5′ GGAAGTAAAAGTCGTAACAAGG 3′
ITS4 5′ TCCTCCGCTTATTGATATGC 3′
NL1 5′ GCATATCAATAAGCGGAGGAAAAG 3′
NL4 5′ GGTCCGTGTTTCAAGACGG 3′
Reference of primer
  • White, T. J., Bruns, T., Lee, S., and Taylor, J. W. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, In: Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J. (eds.), PCR Protocols: A Guide to Methods and Applications, pp. 315-322, Academic Press, New York.
  • O’Donell, K. (1993). Fusarium and its near relatives, In: Reynolds, D. R. and Taylor, J. W. (eds.), The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics, pp. 225-233, CAB International, Wallingford.

Related Website

Related Articles



 

コメントは受け付けていません。