Latest revision as of 19:24, 9 September 2010

Materials

You will need:

Sterilized milliQ water
BSA
NEB buffers 1,2,3,and/or 4 (see extra notes)
Miniprepped sample or plasmid you wish to cut
NEB digestion enzymes (EcoRI, SpeI, PstI, NheI, and/or XbaI)

Extra Notes

Be sure to use the buffer that maximizes the compatibility and activity between the two enzymes. The numbers indicate the percentage of enzyme activity for each enzyme in each buffer.

Enzyme	Buffer 1	Buffer 2	Buffer 3	Buffer 4
EcoRI	100	100	100	100
SpeI	75	100	25	100
PstI	75	75	100	50
NheI	100	100	10	100
XbaI	0	100	75	100

Procedure

1. Add the following into a PCR tube

22μL of milliQ water
1μL BSA
5μL buffer x (see extra notes)
20μL template to be cut
1μL digestion enzyme 1
1μL digestion enzyme 2

2. Run in a thermocycler

37°C for 3 hours
80°C for 20 minutes
Keep at 4°C if to be stored

Purpose

To cut DNA at the designated places; cut out the insert from the plasmid.

References


Materials Extra Notes Procedure Purpose References

We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!

We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)

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-. Run in a machine
+. Run in a thermocycler
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 <li>37°C for 3 hours </li>
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 <li><a href="#materials" class="help">Materials</a></li>
+<li><a href="#extranotes" class="help">Extra Notes</a></li>
 <li><a href="#procedure" class="help">Procedure</a></li>
 <li><a href="#purpose" class="help">Purpose</a></li>

Team:UC Davis/protocols/digestion.html

From 2010.igem.org