McClean: Yeast Recombinational Cloning

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#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#Anecdotal observations that might be of use to others can also be posted here.   
#Anecdotal observations that might be of use to others can also be posted here.   
 +
 +
'''*[[User:Megan N McClean|Megan N McClean]] 19:24, 20 April 2012 (EDT)''': This is my rough outline of the protocol I have been using.  Many things need to be clarified, so please feel free to do so as you perfect this protocol yourself.  Things to add:
 +
* Approximate concentrations of DNA (PCR product and vector) that I have been using
 +
* Some strains and vector backbones that have worked together successfully
 +
* Optimized conditions for getting the plasmids back into bacteria (the hardest part so far)
 +
* Some examples of primer designs that have been successful
 +
* If anyone does this with multiple fragments (ie, to also combine 1 or more fragments before they are inserted into the backbone) please add that to the protocol
 +
 +
 +
'''*[[User:Michael T. Patel|Michael T. Patel]] 13:16, 20 May 2013 (EDT)''': Some observations/additions:
 +
* I typically transform DNA in a 2:1 ratio of PCR insert to digested vector. I usually eyeball the relative concentration by running equal volumes of each on a gel. I never use more than 10 μL of PCR product and 5 μL of vector.
 +
* Unless I'm going to use it for other purposes, I don't use the Qiagen purification kits to purify the digested vector. I just kill the enzyme according to NEB directions (usually just a heat-kill step)
 +
* I usually use yMM1146 (it has three common auxotrophies and I usually have it on hand) and any of the Sikorki-Hieter CEN plasmids (pMM5-8).
 +
* When I do this with multiple fragments, I maintain the above ratio with each fragment. So if I use 2 μL of vector, I'll use 4 μL of each fragment (assuming roughly equal concentration.)
 +
* Because we usually eyeball concentrations, it is probably helpful to plate at least 2 different concentrations of cells after transformation.
 +
* I also transform a control with only digested vector (no insert.) If your vector + insert plate has about 10 fold more colonies than the vector control, then your cloning likely worked.
 +
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
==References==
==References==
-
Adapted from the Goode Lab protocol: [http://www.bio.brandeis.edu/goodelab/ The Goode Lab at Brandeis University]
 
==Contact==
==Contact==

Current revision


Contents

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

  1. 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)
  2. PCR up your insert of interest using your primers with appropriate homology to the vector backbone.
  3. 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)
  4. Pick successful colonies from the yeast transformation and prep plasmid from them using the Zymoprep Yeast Plasmid Miniprep II protocol
  5. Transform the prepped plasmid into bacteria
  6. Purify plasmid DNA from successful bacterial transformants and verify the insert (sequencing, RE digest, etc).

Notes

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

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

*Megan N McClean 19:24, 20 April 2012 (EDT): This is my rough outline of the protocol I have been using. Many things need to be clarified, so please feel free to do so as you perfect this protocol yourself. Things to add:

  • Approximate concentrations of DNA (PCR product and vector) that I have been using
  • Some strains and vector backbones that have worked together successfully
  • Optimized conditions for getting the plasmids back into bacteria (the hardest part so far)
  • Some examples of primer designs that have been successful
  • If anyone does this with multiple fragments (ie, to also combine 1 or more fragments before they are inserted into the backbone) please add that to the protocol


*Michael T. Patel 13:16, 20 May 2013 (EDT): Some observations/additions:

  • I typically transform DNA in a 2:1 ratio of PCR insert to digested vector. I usually eyeball the relative concentration by running equal volumes of each on a gel. I never use more than 10 μL of PCR product and 5 μL of vector.
  • Unless I'm going to use it for other purposes, I don't use the Qiagen purification kits to purify the digested vector. I just kill the enzyme according to NEB directions (usually just a heat-kill step)
  • I usually use yMM1146 (it has three common auxotrophies and I usually have it on hand) and any of the Sikorki-Hieter CEN plasmids (pMM5-8).
  • When I do this with multiple fragments, I maintain the above ratio with each fragment. So if I use 2 μL of vector, I'll use 4 μL of each fragment (assuming roughly equal concentration.)
  • Because we usually eyeball concentrations, it is probably helpful to plate at least 2 different concentrations of cells after transformation.
  • I also transform a control with only digested vector (no insert.) If your vector + insert plate has about 10 fold more colonies than the vector control, then your cloning likely worked.


Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Contact

or instead, discuss this protocol.

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