Double Digest Guide

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===iGEM Double Digest Guide=== by Karmella Haynes, 2006
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<font size="5">iGEM Double Digest Guide</font><br>by Karmella Haynes, 2006
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{| width="800px" cellspacing="0"
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{| width="700px" cellspacing="0" cellspadding="5"
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|- valign="top"
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| colspan="4" | '''Standard BioBrick Cloning Sites''' (Knight)
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|- valign="top"
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| colspan="4" style="background:lightgrey"|<font face="courier">5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG--<br>3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC--</font>
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|- valign="top"
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| colspan="4" style="background:lightgrey"|<font face="courier"><font color="lightgrey">5'--<font color="black">EcoRI</font>- --<font color="black">NotI</font>-- - -<font color="black">XbaI</font>- - -------------- T -<font color="black">SpeI</font>- - -<font color="black">NotI</font>-- -<font color="black">PstI</font>---</font>
|- valign="top"
|- valign="top"
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| style="width:180px" | '''Standard BioBrick Cloning Sites''' (Knight)
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| width="100" | '''Enzymes'''
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| style="background:lightgrey"|<font face="courier">5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG--<br>3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC--</font>
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| width="150" | '''Buffer'''
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| width="100" | '''Temperature'''
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| '''Purpose'''
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|- valign="top"
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| style="width:180px" | <font color="white">...</font>
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| EcoRI, XbaI || Low || 37&#186;C || To create a "Front Vector"
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| style="background:lightgrey"|<font face="courier"><font color="lightgrey">5'--<font color="black">EcoRI</font>- --<font color="black">NotI</font>-- - -<font color="black">XbaI</font>- - -------------- T -<font color="black">SpeI</font>- - -<font color="black">NotI</font>-- -<font color="black">PstI</font>---</font>
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|- valign="top"
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|}
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| EcoRI, SpeI || Low || 37&#186;C || To create a "Front Insert"
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|- valign="top"
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| SpeI, PstI || Medium || 37&#186;C || To create a "Back Vector"
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|- valign="top"
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| XbaI, PstI || Low || 37&#186;C || To create a "Back Insert"
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|- valign="top"
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| EcoRI, PstI || Promega&#174; Buffer H || 37°C || To excise entire insert or validate part size/ restriction sites
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|- valign="top"
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| XbaI, SpeI || Low || 37&#186;C || To excise entire insert, validate part size/ restriction sites, or clone a PCR with inverted sites
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{|style="width:700px"
 
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|-
 
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| '''Enzymes''' || '''Buffer''' || '''Temperature''' || '''Purpose'''
 
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|-
 
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| EcoRI, XbaI || Low || 37°C || To create a "Front Vector"
 
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|-
 
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| EcoRI, SpeI || Low || 37°C || To create a "Front Insert"
 
-
|-
 
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| SpeI, PstI || Medium || 37°C || To create a "Back Vector"
 
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|-
 
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| XbaI, PstI || Low || 37°C || To create a "Back Insert"
 
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|-
 
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| EcoRI, PstI || Promega® Buffer H || 37°C || To excise entire insert or validate part size
 
|}
|}
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'''Davidson Buffers''' [10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mg/mL BSA, X mM NaCl]
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'''Davidson Buffers''' [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
* '''0 (zero)''', 0 NaCl
* '''Low''', 50 mM NaCl
* '''Low''', 50 mM NaCl
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* '''High''', 150 mM NaCl
* '''High''', 150 mM NaCl
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'''Promega® Buffer H''' [90 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl]
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'''Promega&#174; Buffer H''' [90 mM Tris-HCl pH 7.5, 10 mM MgCl<sub>2</sub>, 50 mM NaCl]
===References===
===References===
* [https://dspace.mit.edu/handle/1721.1/21168:Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks]
* [https://dspace.mit.edu/handle/1721.1/21168:Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks]

Latest revision as of 22:07, 7 November 2006

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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