Double Digest Guide
From 2006.igem.org
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- | | 5' || --gca || GAATTC || GCGGCCGC || T || TCTAGA || G || --insert-- || T || ACTAGT || A || GCGGCCG || CTGCAG || gct-- | + | | 5' || <code>--gca</code> || <code>GAATTC</code> || <code>GCGGCCGC</code> || T || TCTAGA || G || --insert-- || T || ACTAGT || A || GCGGCCG || CTGCAG || gct-- |
|- | |- | ||
| || --cgt || CTTAAG || CGCCGGCG || A || AGATCT || C || ------------ || A || TGATCA || T || CGCCGGC || GACGTC || cga-- | | || --cgt || CTTAAG || CGCCGGCG || A || AGATCT || C || ------------ || A || TGATCA || T || CGCCGGC || GACGTC || cga-- |
Revision as of 16:06, 14 September 2006
iGEM Double Digest Guide by Karmella Haynes, 2006
5' | --gca | GAATTC | GCGGCCGC | T | TCTAGA | G | --insert-- | T | ACTAGT | A | GCGGCCG | CTGCAG | gct-- |
--cgt | CTTAAG | CGCCGGCG | A | AGATCT | C | ------------ | A | TGATCA | T | CGCCGGC | GACGTC | cga-- | |
EcoRI | NotI | XbaI | 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]