Double Digest Guide
From 2006.igem.org
(Difference between revisions)
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| - | '''iGEM Double Digest Guide''' | + | '''iGEM Double Digest Guide''' by Karmella Haynes, 2006 |
| - | by Karmella Haynes, 2006 | + | |
| - | + | {|style="background:silver" | |
| - | 5' | + | |- |
| - | --gca GAATTC GCGGCCGC T TCTAGA G --insert-- T ACTAGT A GCGGCCG CTGCAG gct-- | + | | 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 || | ||
| + | |} | ||
| - | |||
| - | |||
| - | {| | + | {|style="width:700px" |
|- | |- | ||
| '''Enzymes''' || '''Buffer''' || '''Temperature''' || '''Purpose''' | | '''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 || 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] | ||
Revision as of 21:03, 13 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 | Buffer H* | 37°C | To excise entire insert or validate part size |
| 0 (zero) | 0 NaCl |
| Low | 50 mM NaCl |
| Medium | 100 mM NaCl |
| High | 150 mM NaCl
|
