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œœœœœ@TAB restriction buffer@œœœœœ



TAB buffer is a general restriction enzyme buffer. Because it works pretty well with almost all restriction enzymes (including SmaI & other KCL-prefering enzymes), it makes double & triple digests particularly easy. This is essentially the buffer recommended by Maniatis for use in blunting 3' or 5' overhanging ends with T4 DNA polymerase.

  1. Materials

    1. 1M Tris-OAc, pH 7.9
      12.1g Tris base, adjusted to pH 7.9 with glacial acetic acid, per 100ml

    2. 1M KOAc (potassium acetate)

    3. 1M MgOAc (magnesium acetate)

    4. 50mg/ml nuclease-free BSA

    5. 1M dithiothreitol

    6. 5X TAB is prepared by mixing:
      1.65ml 1M Tris-OAc, pH7.9
      3.3ml 1M KOAc
      0.5ml 1M MgOAc
      0.1ml 50mg/ml BSA
      4.45ml ddH2O
      Store this "pre-mix" frozen in 1ml aliquots
      As each 1ml tube is brought out for use, add 5ul 1M DTT. This is now the complete 5X TAB buffer. Store at 4C.

    7. Use as you would any restriction enzyme buffer, except:

      1. The concentrated stock is 5X rather than 10X.
      2. Multiple restriction digests are done simultaneously.
      3. Because most enzymes don't give quite full activity in TAB, use a little extra enzyme. This is not generally a problem, since most restriction enzymes today are sold at 5-20 units/ul & most people use much more than 1 unit/ug DNA already.


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