McClean: Designing "Yeast Toolkit" compatible primers
Overview
This protocol is written to prepare the user to design PCR primers such that the end product will have BsaI and BsmBI cut sites, as well as the correct flanking overhangs. It is intended to supplement both Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology at <http://pubs.acs.org/doi/pdf/10.1021/sb500366v> (2015) and its own supplementary information.
Materials
- NCBI Primer-Blast (or other primer design tool)
- Basic understanding of Golden Gate Assembly (search for an overview in other websites if link doesn't work)
- An understanding of what DNA sequences BsaI and BsmBI recognize and where they cut.
Protocol
- Design primers as normal, such that they can be used to PCR amplify your gene of interest. NCBI's Primer-Blast tool is handy for this. See McClean: designing primers for more help.
- Prefix the 5' end of your forward primer with GCATCGTCTCATCGGTCTCANNNN
- Prefix the 5' end of your reverse primer with ATGCCGTCTCAGGTCTCANNNN
- The desired end product will be this, where NNNN is the type specific overhang and can be found in the paper's SI.
- Forward primer --->
- 5'- GCATCGTCTCATCGGTCTCANNNN- gene of interest - NNNNTGAGACCTGAGACGGCAT -3'
- 3'- CGTAGCAGAGTAGCCAGAGTNNNN- gene of interest - NNNNACTCTGGACTCTGCCGTA -5'
- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . <--- Reverse Primer
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- 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.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology at <http://pubs.acs.org/doi/pdf/10.1021/sb500366v> (2015)
Contact
- Cameron J. Stewart 12:22, 15 September 2016 (EDT):
or instead, discuss this protocol.
