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Team:LMU-Munich/Notebook/Protocols/5 Restriction digest
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(New page: {{:Team:LMU-Munich/Templates/Page Header}} Restrictionsdigest (Source: http://www.promega.com/enotes/applications/ap0078.htm, http://openwetware.org/wiki/Engineering_BioBrick_vectors_fr...) |
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Table 1. Restrictionenzymes with data about their activity in selected buffers. Optimal also taken from following webpage especially if double digestion wanted: http://www.promega.com/guides/re_guide/research.asp?search=buffer | Table 1. Restrictionenzymes with data about their activity in selected buffers. Optimal also taken from following webpage especially if double digestion wanted: http://www.promega.com/guides/re_guide/research.asp?search=buffer | ||
| - | + | <table> | |
| - | Enzyme Activity in buffer A Activity in buffer B Activity in buffer C Activity in buffer D Activity in buffer H Activity in buffer MC | + | <tr><th>Enzyme</th> <th>Activity in buffer A</th> <th>Activity in buffer B</th> <th>Activity in buffer C</th> <th>Activity in buffer D</th> <th>Activity in buffer H</th> <th>Activity in buffer MC</th></tr> |
| - | XbaI 50-75 75-100 75-100 100 - 100 | + | <tr> <td>XbaI</td> <td>50-75</td> <td>75-100</td> <td>75-100</td> <td>100</td> <td>-</td> <td>100</td></tr> |
| - | EcoRI 25-50 50-75 50-75 50-75 100 100 | + | <tr><td>EcoRI</td> <td>25-50</td> <td>50-75</td> <td>50-75</td> <td>50-75</td> <td>100</td> <td>100</td></tr> |
| - | PstI 10-25 50-75 50-75 50-75 100 25-50 | + | <tr><td>PstI</td> <td>10-25</td> <td>50-75</td> <td>50-75</td> <td>50-75</td> <td>100</td> <td>25-50</td></tr> |
| - | SpeI 75-100 100 75-100 75-100 - 100 | + | <tr><td>SpeI</td> <td>75-100</td> <td>100</td> <td>75-100</td> <td>75-100</td> <td>-</td> <td>100</td></tr> |
| + | </table> | ||
Latest revision as of 17:44, 11 August 2010
(Source: http://www.promega.com/enotes/applications/ap0078.htm, http://openwetware.org/wiki/Engineering_BioBrick_vectors_from_BioBrick_parts/Restriction_digest)
plasmid DNA 1µg
restriction enzyme 1µl
10X buffer 2µl
BSA (10µg/µl) 0.2µl
nuclease-free water to final volume 20µl
Table 1. Restrictionenzymes with data about their activity in selected buffers. Optimal also taken from following webpage especially if double digestion wanted: http://www.promega.com/guides/re_guide/research.asp?search=buffer
Vortex all reagents before use. (especially the enzymes)
1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
2. Add restriction enzyme buffer.
3. Add BSA.
4. Add DNA.
5. Add each enzyme.
Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
6. Incubate for 2 hours at 37°C.
7. Incubate for 20 mins at 80°C to heat inactivate enzyme.
This step is sufficient to inactivate even Pst I.
8. Incubate 4°C until you pull the reaction out of the thermal cycler. (Thermal cycler can be used for a installed program for digestion, even overnight)
Restrictionsdigest
Restriction enzyme digestions were assembled as follows:
Enzyme Activity in buffer A Activity in buffer B Activity in buffer C Activity in buffer D Activity in buffer H Activity in buffer MC
XbaI 50-75 75-100 75-100 100 - 100 EcoRI 25-50 50-75 50-75 50-75 100 100 PstI 10-25 50-75 50-75 50-75 100 25-50 SpeI 75-100 100 75-100 75-100 - 100
Procedure
