Revision as of 18:00, 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 of the enzyme activity between the two enzymes.

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 machine

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 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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Team:UC Davis/protocols/digestion.html

From 2010.igem.org