McClean: Yeast Recombinational Cloning: Difference between revisions

Revision as of 16:21, 20 April 2012

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

Digest plasmid following NEB protocol appropriate to the enzyme that you are using. Clean-up the product (usually I just use the Qiagen PCR purification kit)
PCR up your insert of interest using your primers with appropriate homology to the vector backbone.
Transform the digested plasmid and the PCR'd insert into the appropriate yeast strain. Usually I transform about 28μL of PCR product and 7μL of vector (and yes, so far I have been successful without measuring the actual concentrations of either of these)
Pick successful colonies from the yeast transformation and prep plasmid from them using the Zymoprep Yeast Plasmid Miniprep II protocol
Transform the prepped plasmid into bacteria
Purify plasmid DNA from successful bacterial transformants and verify the insert (sequencing, RE digest, etc).

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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Adapted from the Goode Lab protocol: The Goode Lab at Brandeis University

@@ Line 22: / Line 22: @@
 ==Protocol==
-# D
+# Digest plasmid following NEB protocol appropriate to the enzyme that you are using.  Clean-up the product (usually I just use the Qiagen PCR purification kit)
+# PCR up your insert of interest using your primers with appropriate homology to the vector backbone.
+# Transform the digested plasmid and the PCR'd insert into the appropriate yeast strain.  Usually I transform about 28μL of PCR product and 7μL of vector (and yes, so far I have been successful without measuring the actual concentrations of either of these)
+# Pick successful colonies from the yeast transformation and prep plasmid from them using the Zymoprep Yeast Plasmid Miniprep II protocol
+# Transform the prepped plasmid into bacteria
+# Purify plasmid DNA from successful bacterial transformants and verify the insert (sequencing, RE digest, etc).
 ==Notes==
 <!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->