Difference between revisions of "Help:Protocols/Restriction Digest"
m
Line 1: Line 1:
−
At iGEM HQ we use this protocol for restriction digests along with enzymes purchased from NEB.
+
__NOTOC__
 +
{{HelpPage/MainCSS}}
 +
<html>
 +
<style type="text/css">
 +
#mm-help img {opacity: 1}
  
 +
</style>
 +
 +
<table id="nav-table">
 +
<tr><td colspan="3"></html>{{HelpPage/MiniMap}}<html></td></tr>
 +
<tr>
 +
<td style="width: 120px; height: 100px;" rowspan="2"></td>
 +
<td  colspan="2"><div id="splash-title">Restriction Digests</div></td>
 +
</tr>
 +
<tr>
 +
<td colspan="2" style="vertical-align:text-top;">
 +
When using parts for 3A assembly, or testing the quality of a part you'll need to run a restriction digest.
 +
</td>
 +
</tr>
 +
</table>
 +
 +
</html>
 +
 +
{{TOCright}}
 +
At iGEM HQ we recommend using the following protocols. The large reaction protocol will have enough of a volume for ligation and you'll be able to use a portion to run a gel. The small reaction protocol will only have enough volume for 
 
==Materials==  
 
==Materials==  
 
*PCR tube
 
*PCR tube
Line 19: Line 42:
 
7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.<br> ''We incubate in a thermocycler with a heated lid''<br>
 
7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.<br> ''We incubate in a thermocycler with a heated lid''<br>
 
8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2ul of the digest (20ng of DNA) for ligations.
 
8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2ul of the digest (20ng of DNA) for ligations.
 +
 +
 +
[[Linearized Plasmid Backbones Original Protocol]]
 +
 +
 +
{{HelpPage/Footer}}

Revision as of 14:43, 17 May 2012

At iGEM HQ we recommend using the following protocols. The large reaction protocol will have enough of a volume for ligation and you'll be able to use a portion to run a gel. The small reaction protocol will only have enough volume for

Materials

  • PCR tube
  • dH20
  • Enzymes (EcoRI, XbaI, SpeI, PstI)
  • BSA
  • Enzyme Buffer (NEBuffer 2)*

Notes: You should keep all materials on ice.

Protocol

1. Add 500ng of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.
2. Add 5ul of NEBuffer 2 to the tube.
3. Add 0.5ul of BSA to the tube.
4. Add 1ul of your first enzyme.
5. Add 1ul of your second enzyme.
6. There should be a total volume of 50ul. Mix well and spin down.
7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.
We incubate in a thermocycler with a heated lid
8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2ul of the digest (20ng of DNA) for ligations.


Linearized Plasmid Backbones Original Protocol