Double Digest Guide
From 2006.igem.org
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]
