Double Digest Guide
From 2006.igem.org
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]
