Double Digest Guide

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	{|style="background:silver"		{|style="background:silver"
	|-		|-
-	| 5' || --gca || GAATTC || GCGGCCGC || T || TCTAGA || G || --insert--  || T || ACTAGT || A || GCGGCCG || CTGCAG || gct--	+	| [[Image:BioBricks_from_paper.png]]
-	|-	+
-	|    || --cgt || CTTAAG || CGCCGGCG || A || AGATCT || C || ------------ || A || TGATCA || T || CGCCGGC || GACGTC || cga--	+
-	|-	+
-	|    ||      || EcoRI  || NotI    ||  || XbaI  ||  ||              ||  || SpeI  ||  || NotI    || PstI  ||	+
	|}		|}

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Revision as of 23:38, 19 September 2006

iGEM Double Digest Guide by Karmella Haynes, 2006

File:BioBricks from paper.png

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	Promega® 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]