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Team:Grenoble/Biology/Protocols/Restriction
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<h1>Restriction enzyme digestion of DNA</h1> | <h1>Restriction enzyme digestion of DNA</h1> | ||
<h2>Goal</h2> | <h2>Goal</h2> | ||
| - | Digest DNA in order to perform a ligation or to check a construction. | + | Digest DNA in order to perform a <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Ligation">ligation</a> or to check a construction. |
</section> | </section> | ||
<br/> | <br/> | ||
| + | <section> | ||
| + | <h2>Enzymes</h2> | ||
| + | <ul><li><a href="http://www.neb.com/nebecomm/products/productR0145.asp" target="_blank">XbaI</a></li> | ||
| + | <li><a href="http://www.neb.com/nebecomm/products/productR0133.asp" target="_blank">SpeI</a></li> | ||
| + | <li><a href="http://www.neb.com/nebecomm/products/productR0140.asp" target="_blank">PstI</a></li> | ||
| + | <li><a href="http://www.neb.com/nebecomm/products/productR0101.asp" target="_blank">EcoRI</a></li> | ||
| + | </section> | ||
| + | |||
<section> | <section> | ||
<h2>Protocol</h2> | <h2>Protocol</h2> | ||
| Line 17: | Line 25: | ||
<li>In an eppendorf tube, introduce : <ul><div class="petit"> | <li>In an eppendorf tube, introduce : <ul><div class="petit"> | ||
<li>2 µL of each restriction enzyme</li> | <li>2 µL of each restriction enzyme</li> | ||
| - | <li>10 µL of | + | <li>10 µL of NEB 2 buffer</li> |
| - | <li>1 µL of Bovine Serum Albumin (BSA) | + | <li>1 µL of Bovine Serum Albumin (BSA)</li> |
<li>variable volume of template DNA</li> | <li>variable volume of template DNA</li> | ||
<li>to 50 µL of water</li> | <li>to 50 µL of water</li> | ||
| Line 25: | Line 33: | ||
<br/> | <br/> | ||
<center> | <center> | ||
| - | <table> | + | <table class="tableau"> |
<tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr> | <tr><th><i><div class="petit"><center>SAFETY AND USEFUL RECOMMANDATION</center></div></i></th></tr> | ||
<tr><td><div class="petit">Those products are not dangerous at first sight, but in case of spreading | <tr><td><div class="petit">Those products are not dangerous at first sight, but in case of spreading | ||
| - | or | + | or contacts with any part of the body, it is recommended to wash this part and |
| - | to dry it after. | + | to dry it after. Use a different pipette tip for each sample. It is also important to |
| - | + | close tubes after putting the different reagents. DNA is more sensitive to its environment while it is not circulared in a plasmid and introduced | |
| - | + | into a cell. Therefore, a spreading has very few chances to lead to any bad consequences.</div></td></tr> | |
| - | + | ||
</table> | </table> | ||
</center> | </center> | ||
Latest revision as of 08:20, 25 September 2012
Restriction enzyme digestion of DNA
Goal
Digest DNA in order to perform a ligation or to check a construction.Enzymes
Protocol
- Set up the water bath at 37°C.
- In an eppendorf tube, introduce :
- 2 µL of each restriction enzyme
- 10 µL of NEB 2 buffer
- 1 µL of Bovine Serum Albumin (BSA)
- variable volume of template DNA
- to 50 µL of water
SAFETY AND USEFUL RECOMMANDATION Those products are not dangerous at first sight, but in case of spreading or contacts with any part of the body, it is recommended to wash this part and to dry it after. Use a different pipette tip for each sample. It is also important to close tubes after putting the different reagents. DNA is more sensitive to its environment while it is not circulared in a plasmid and introduced into a cell. Therefore, a spreading has very few chances to lead to any bad consequences.
- Put the eppendorf tube into the water bath.
- Incubate at 37°C for 15 minutes.
