McClean: Yeast Recombinational Cloning
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Overview
This is a protocol for inserting a piece of DNA into a plasmid using homologous recombination in yeast. It works roughly as follows:
- a plasmid (CEN or 2-micron with selectable yeast and bacterial markers) is digested with a restriction enzyme somewhere in the middle of where you would like the DNA to insert into the plasmid
- the fragment to insert is PCR amplified with primers that also contain 40bp of homology to the plasmid, such that when the PCR product is recombined into the plasmid at the sites homology you get everything in the desired locations
- the DNA and digested plasmid are simultaneously transformed into a yeast strain that allows for selection for the recombined plasmid (ie, if your plasmid contains a LEU2 marker, transform into a leu- strain)
- DNA is prepped from several transformants (using Zymo Research's Zymoprep Yeast Plasmid Miniprep II kit) and transformed into competent 'e. coli' cells
- plasmid DNA is prepped from successful 'e. coli' transformants and sent for sequencing
Materials
- Primers (to amplify the insert) with appropriate homology to the plasmid
- Plasmid that you would like to combine insert into
- PCR reagents (see:
- Appropriate yeast strain (such that you can select for plasmid recombination)
- Reagents for tranformation of yeast (see:
- Zymoprep Yeast Plasmid Miniprep Kit II (Zymo Research)
- Reagents for transformation of competent 'e.coli' cells (see:
Protocol
- D
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
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- Anecdotal observations that might be of use to others can also be posted here.
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References
Adapted from the Goode Lab protocol: The Goode Lab at Brandeis University
Contact
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.
