MaCampbell at 19:28, 6 October 2008

2008-10-06T19:28:17Z

Synthetic Students at 20:03, 11 July 2007

2007-07-11T20:03:34Z

New page

iGEM Double Digest Guide by Karmella Haynes, 2006

{| width="700px" cellspacing="0" cellspadding="5"
|- valign="top"
| colspan="4" | '''Standard BioBrick Cloning Sites''' (Knight)
|- valign="top"
| colspan="4" style="background:lightgrey"|5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG-- 3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC--
|- valign="top"
| colspan="4" style="background:lightgrey"|5'--EcoRI- --NotI-- - -XbaI- - -------------- T -SpeI- - -NotI-- -PstI---
|- valign="top"
| width="100" | '''Enzymes'''
| width="150" | '''Buffer'''
| width="100" | '''Temperature'''
| '''Purpose'''
|- valign="top"
| EcoRI, XbaI || Low || 37ºC || To create a "Front Vector"
|- valign="top"
| EcoRI, SpeI || Low || 37ºC || To create a "Front Insert"
|- valign="top"
| SpeI, PstI || Medium || 37ºC || To create a "Back Vector"
|- valign="top"
| XbaI, PstI || Low || 37ºC || To create a "Back Insert"
|- valign="top"
| EcoRI, PstI || Promega® Buffer H || 37°C || To excise entire insert or validate part size/ restriction sites
|- valign="top"
| 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===
* [https://dspace.mit.edu/handle/1721.1/21168:Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks]

@@ Line 30: / Line 30: @@
-'''Davidson Buffers''' [10 mM Tris-HCl pH 7.5, 10 mM MgCl<sub>2</sub>, 0.1 mg/mL BSA, X mM NaCl]
+'''Davidson Buffers 1X concentrations''' [10 mM Tris-HCl pH 7.5, 10 mM MgCl<sub>2</sub>, 0.1 mg/mL BSA, X mM NaCl]
 * '''0 (zero)''', 0 NaCl
 * '''Low''', 50 mM NaCl

Davidson Missouri W/Double Digest Guide - Revision history

MaCampbell at 19:28, 6 October 2008

Synthetic Students at 20:03, 11 July 2007