Double Digest Guide
From 2006.igem.org
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| + | | style="width:180px" | '''Standard BioBrick Cloning Sites''' (Knight) | ||
| + | | 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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| + | | style="width:180px" | <font color="white">...</font> | ||
| + | | 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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'''Promega® Buffer H''' [90 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl] | '''Promega® Buffer H''' [90 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl] | ||
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===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] | ||
Revision as of 21:49, 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 | 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]
