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Team:Paris Saclay/digestion ligation
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Revision as of 14:46, 4 October 2013 by Thaonguyenlelam (Talk | contribs)
Digestion and Ligation
Digestion :
| Enzyme | EcoRI | PstI | SpeI | XbaI |
|---|---|---|---|---|
|
Buffer |
Fast Digest |
Fast Digest |
Fast Digest |
Fast Digest |
|
Site |
5'-G/AATTC-3' 3'-CTTAA/G-5' |
5'-CTGCA/G-3' 3'-G/ACGTC-5' |
5'-A/CTAGT-3' 3'-TGATC/A-5' |
5'-T/CTAGA-3' 3'-AGATC/T-5' |
Double Digestion :
| DNA | volume |
|---|---|
|
Buffer Fast Digest 10X |
3µL |
|
Enzyme 1 |
1.5 μl |
|
Enzyme 2 |
1.5 μl |
|
H20 |
14µL |
Single Digestion :
| DNA | volume |
|---|---|
|
Buffer Fast Digest 10X |
3µL |
|
Enzyme |
1.5 μl |
|
H20 |
15.5µL |
1. Incubate at 37°C for 10-90min
2. Inactivate digestion of enzymes by ethanol precipitation
Ligation:
Mix in a molar ratio the insert and the linear vector
