Difference between revisions of "Team:Freiburg/Protocols/Restriction"
| Line 21: | Line 21: | ||
<p> | <p> | ||
| − | Material: restriction endonucleases (store on ice), buffer, | + | Material: restriction endonucleases (store on ice), buffer, dH<sub>2</sub>O, heat block or incubator (37°C)<br/> |
| − | Duration: 2 | + | Duration: 2.5 h<br/> |
</p> | </p> | ||
| Line 33: | Line 33: | ||
<h3>Mix</h3> | <h3>Mix</h3> | ||
<table class="tabelle"> | <table class="tabelle"> | ||
| − | <tr><th><center> | + | <tr><th><center>Ingredient</center></th><th><center>Volume / Mass</center></th></tr> |
<tr><td><center>DNA</center></td><td><center>2 - 3 µg</center></td></tr> | <tr><td><center>DNA</center></td><td><center>2 - 3 µg</center></td></tr> | ||
<tr><td><center>Restriction enzyme</center></td><td><center>1 µl each</center></td></tr> | <tr><td><center>Restriction enzyme</center></td><td><center>1 µl each</center></td></tr> | ||
| − | <tr><td><center>BSA 100x ( | + | <tr><td><center>BSA 100x (optional)</center></td><td><center>0.5 µl</center></td></tr> |
| − | <tr><td><center>NEB buffer | + | <tr><td><center>NEB buffer 10x</center></td><td><center>5 µl</center></td></tr> |
| − | <tr><td><center>dH<sub>2</sub>O</center></td><td><center> | + | <tr><td><center>dH<sub>2</sub>O</center></td><td><center>ad 50 µl</center></td></tr> |
</table> | </table> | ||
| − | Incubate at 37 °C for at least 2 h. <i>If there are more than 2 | + | Incubate at 37 °C for at least 2 h. <i>If there are more than 2 recognition sites per enzyme, incubate for more than 2 h!</i> |
</body> | </body> | ||
Revision as of 19:48, 16 September 2015
Restriction digest
Restriction enzymes are a basic component of classical cloning methods. They have the ability to induce DNA double strand breaks at specific sites leaving a characteristic overhang. By cutting two sequences with the same enzyme and thus producing compatible overhangs, these can be assembled in an easy way. DNA ligases can then be used to covalently link the fragments.
Restriciton digests are commonly used for two purposes. One is extract a desired fragment out of a whole plasmid to further use it for subcloning purposes (preparative digest). Another possibility to make use of a restriction digest is to verify successful insertion of a fragment by comparing the resulting fragments to theoretical expectations (analytical digest).
DNA Restriction Digest
Material: restriction endonucleases (store on ice), buffer, dH2O, heat block or incubator (37°C)
Duration: 2.5 h
The amount of DNA that is used for a reaction is dependent on the purpose. For an analytical digest, about 500 ng of DNA are sufficient to visualize the result on an agarose gel. If you aim to extract the DNA after agarose gel analysis, 2 - 3 µg of DNA can be used to obtain high yields.
Mix
