Double Digest Guide
From 2006.igem.org
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| + | | colspan="4" | '''Standard BioBrick Cloning Sites''' (Knight) | ||
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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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| + | | 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> | ||
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| - | + | | width="100" | '''Enzymes''' | |
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| width="150" | '''Buffer''' | | width="150" | '''Buffer''' | ||
| - | | '''Temperature''' | + | | width="100" | '''Temperature''' |
| '''Purpose''' | | '''Purpose''' | ||
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| - | | EcoRI, XbaI || Low || | + | | EcoRI, XbaI || Low || 37ºC || To create a "Front Vector" |
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| - | | EcoRI, SpeI || Low || | + | | EcoRI, SpeI || Low || 37ºC || To create a "Front Insert" |
|- valign="top" | |- valign="top" | ||
| - | | SpeI, PstI || Medium || | + | | SpeI, PstI || Medium || 37ºC || To create a "Back Vector" |
|- valign="top" | |- valign="top" | ||
| - | | XbaI, PstI || Low || | + | | XbaI, PstI || Low || 37ºC || To create a "Back Insert" |
|- valign="top" | |- valign="top" | ||
| - | | EcoRI, PstI || | + | | EcoRI, PstI || Promega® Buffer H || 37°C || To excise entire insert or validate part size/ restriction sites |
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| - | | XbaI, SpeI || Low || 37 º C || To excise entire insert, validate part size/ restriction sites, or clone a PCR with inverted sites | + | | XbaI, SpeI || Low || 37ºC || To excise entire insert, validate part size/ restriction sites, or clone a PCR with inverted sites |
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| - | '''Davidson Buffers''' [10 mM Tris-HCl pH 7.5, 10 mM | + | '''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 | ||
| - | ''' | + | '''Promega® 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]
