Team:UC Davis/protocols/digestion.html

From 2010.igem.org

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  <td>Enzyme</td>
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Revision as of 17:52, 9 September 2010

Digestion

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

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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