Davidson:Double Digest Guide

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iGEM Double Digest Guide
by Karmella Haynes, 2006


Standard BioBrick Cloning Sites (Knight)
5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG--
3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC--
5'--EcoRI- --NotI-- - -XbaI- - -------------- T -SpeI- - -NotI-- -PstI---
Enzymes Buffer Temperature Purpose
EcoRI, XbaI Low 37ºC To create a "Front Vector"
EcoRI, SpeI Low 37ºC To create a "Front Insert"
SpeI, PstI Medium 37ºC To create a "Back Vector"
XbaI, PstI Low 37ºC To create a "Back Insert"
EcoRI, PstI Promega® Buffer H 37°C To excise entire insert or validate part size/ restriction sites
XbaI, SpeI Low 37ºC To excise entire insert, validate part size/ restriction sites, or clone a PCR with inverted sites


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]

References

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