Team:WashU/Protocols/Digestion

From 2012.igem.org

(Difference between revisions)
Line 1: Line 1:
 +
<!--Thanks to NEB for the above instructions.  The original source can be found here: http://www.neb.com/nebecomm/products/protocol445.asp-->
 +
{{WashUback}}
{{WashUback}}
Line 16: Line 18:
4.Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.
4.Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.
-
 
-
==Source==
 
-
Thanks to NEB for the above instructions.  The original source can be found here: [http://www.neb.com/nebecomm/products/protocol445.asp]
 

Revision as of 19:50, 19 June 2012



General Digestion Protocol

1.Digest Upstream Part with EcoRI-HF™ and SpeI.

Add 500 ng of plasmid DNA, EcoRI-HF (1 µl), Spel (1 µl), 10X NEBuffer 2 (5 µl), 100X BSA (0.5 µl),H2O to total 50 µl

2.Digest Downstream Part with XbaI and PstI.

Add 500 ng of plasmid DNA, XbaI (1 µl), PstI (1 µl), 10X NEBuffer 2 (5 µl), 100X BSA (0.5 µl),H2O to total 50 µl

3.Digest the Destination Plasmid with EcoRI-HF and PstI. The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.

Add 500 ng of plasmid DNA, EcoRI-HF (1 µl), PstI (1 µl), 10X NEBuffer 2 (5 µl), 100X BSA (0.5 µl),H2O to total 50 µl

4.Incubate all three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Retrieved from "http://2012.igem.org/Team:WashU/Protocols/Digestion"