Davidson Missouri W/Double Digest Guide

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 1X concentrations [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

• Tom. Idempotent Vector Design for Standard Assembly of Biobricks