Double Digest Guide

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iGEM Double Digest Guide by Karmella Haynes, 2006

5' --gca GAATTC GCGGCCGC T TCTAGA G --insert-- T ACTAGT A GCGGCCG CTGCAG gct--
--cgt CTTAAG CGCCGGCG A AGATCT C ------------ A TGATCA T CGCCGGC GACGTC cga--
EcoRI NotI XbaI SpeI NotI PstI


Enzymes Buffer Temperature Purpose
EcoRI, XbaI Low 37°C To create a "Front Vector"
EcoRI, SpeI Low 37°C To create a "Front Insert"
SpeI, PstI Medium 37°C To create a "Back Vector"
XbaI, PstI Low 37°C To create a "Back Insert"
EcoRI, PstI Buffer H* 37°C To excise entire insert or validate part size


Davidson Buffers [10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mg/mL BSA, X mM NaCl]
0 (zero) 0 NaCl
Low 50 mM NaCl
Medium 100 mM NaCl
High 150 mM NaCl
  • Promega® Buffer H [90 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl]
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