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( DNA, ).

- , .

  1. (-70o) ;
  2. 37o, ON;
  3. (10-12) ~2-3mm 40ml SOB 0.5-1.0L.
  4. (200-300rpm)
  5. * 37o - , ,
    * 18o ( ) - , - ;

  6. OD600= 0.6

  7. .

  8. 50ml - 30ml ;
  9. 0oC, 10-15';
  10. 2-3krpm 4oC, 10', ;
  11. 2-3krpm 4oC, 20'', ;
  12. 10ml TB;
  13. 0o, 10-15';
  14. .7, 8;
  15. 2ml TB;
  16. + DMSO 3.5% (70µl);
  17. 0o, 10-15';
  18. .14,15;
  19. 100µl 2ml ;
  20. ;
  21. () -70o.
  22. 100 (0.5µg/ml) pDNA 5ng/ml;
  23. 0.5M bMeEtOH ;
  24. :
  25. 4µl 0.5M bMeEtOH,
    2µl (10pg) pDNA;

  26. 0oC, 20-60';
  27. 42oC, 30'', ;
  28. , 2';
  29. + 400µl SOC;
  30. , 37o, 30';
  31. 50µl ;
  32. "N" , = N106[/µg].
    1. "";
    2. pDNA <10µl;
    3. - :
    4. * (, - ), 100-200µl ( - , , (!) ),
      * (, ), 10-50µl (), ON 0o ( ). , .



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    TB

    ( 4o)

    .0.5L1L
    PIPES10mM302.37g/M1.51g3.02g
    MnCl2x2H2O55mM161.9 g/M4.45g8.90g
    MnCl2x4H2O 197.91g/M 5.44g 10.89g
    CaCl2x2H2O15mM147.02 g/M1.10g2.21g
    KCl250mM74.55 g/M9.32g18.64g
    H2O mQ   
  • , MnCl2, KOH pH 6.7(~650 µl 1N 0.5L TB). MnCl2, 0.45µm .
  • PIPES HEPES,:
  • HEPES10mM238.3g/M1.19g2.38g

    SOC, SOB

    ( NT).

    200ml400ml
    Bactotryptone.4.0g8.0g
    Bacto Yest extr..1.1g2.2g
    NaCl5M0.4ml0.8ml
    KCl1M2.0ml4ml
    H2OmQ~196ml~391ml
    1. , <60o;
    2. :
    3. 0.01V 2M Mg2+{= 1M MgCl2+1M MgSO4}   
      0.01V 2M glucose;   

    4. 0.2µm ;
    5. , -20oC, (-70oC).

    SOB , .

    ß-

    0.5M ( +4o).

    .1ml
    ßMeEtOH0.5M14.3M35µl
    H2O mQ965µl

    2M Mg-solution

    ( , NT), p=1.173:

    .100ml
    MgCl2x6H2O1M203.3g/M20.33g
    MgSO4x7H2O1M246.47g/M24.65g
    H2O mQ72.3ml


:
/ /
  • " " "" - .


-- - --

  • : H.Inoue, H.Nojima, H.Okayama "High efficiency transformation of Escherichia coli with plasmids", Gene, 96(1990), 23-8.
  • ~10%.
  • ~10ng pDNA/100µl , .
  • 100-200ng/100µl .
  • >25ng/100µl - .
  • (, ) , (i) (~20µl), (ii) ( "").
  • , DMSO. "Sigma, #D2650", ( 5 10ml). -70o.
  • :
    1. ( DNA, ). - DNA , ..: (i) (ii) E. coli , , .
    2. , -, . ( ) .

    .4, 5.

  • 37o ( ) (A600=0.45 DH10B). 18o A600=0.35-0.70.
  • : OD550=c[cells/ml]*108/k.
  • k OD550
    JM1091.10.36-0.64
    SURE1.60.25-0.44
    XL-1Blue2.00.20-0.35
    XL-1BlueMRF'1.90.21-0.37

    .10.

  • PIPES TB HEPES BES. . pH 5.6-7.0, pH>7.0 MnCl2 .
  • .14.

  • DMSO ( ), . , , " " ( - DMSO ). , ( DMSO ), , .
  • . 17.

  •  , 10 ( , - DNA ).
  • . 18.

  •  ( ) 4-5 .
  •  : ~3-7.5kb;
  • 10kb ~ 50%;
    13kb ~ 27%;
    17.5kb ~ 15%.

    .24.

  • : 40-50o.
  •  , " (1'), ( NT)", ~10 .
: 30/07/13

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