SGA Technology

Protocols

We have made a number of improvements on SGA strains and methods and the following sections describe the latest improvements.

SGA Methodology

We have manuscripts describing SGA methodology:

  • A. Tong and C. Boone (2005)
    Synthetic Genetic Array (SGA) Analysis in Saccharomyces cerevisiae.
    Yeast Protocols, Second Edition. Methods in Molecular Biology, 313:171- 92. (The Humana Press Inc., Totowa, NJ, U. S. A . )
    Download PDF document.
  • A. Tong and C. Boone (2007)
    High-Throughput Strain Construction and Systematic Synthetic Lethal Screening in Saccharomyces cerevisiae.
    Yeast Gene Analysis - Second Edition. Methods in Microbiology, Volume 36: 369-386, 706-707 (Elsevier Ltd.)
    Download PDF document.
  • Freezing down 96 well format libraries.
    Download XLS document.
  • Boone SGA array definition files

SGA Strain Kit

Upon request, we send out an SGA kit that contains: p4339, for switching kanMX deletion alleles to natMX alleles; Y7092, our standard SGA starting strain, into which query mutations are introduced by transformation; Y8205, another SGA starting strain, into which query mutations can be introduced by the switching protocol.

p4339

TA::MX4-natR switcher cassette;  This plasmid can be used to switch from kanR KO kan-MX or URA3 KO URA-MX to natR KO. Cut with Eco RI to target.

P4339 was constructed by amplifying natMX [A. L. Goldstein, J. H. McCusker, Yeast 15, 1541 (1999)] with primers (5'-ACATGGAGGCCCAGAATACCC-3' and 5'-CAGTATAGCGACCAGCATTCAC-3') and the product was introduced into pCRII-TOPO (InVitrogen) at the unique EcoRI sites.

E. coli strain: TOP10F

Plasmid is ampicillin resistant and should be cultured in 2YT + 120ug/ml ampicillin.

Y7092

Starting strain for SGA query strain construction by PCR knockout.

Mating type: MAT alpha
Genotype: can1delta::STE2pr-Sp_his5 lyp1delta his3delta1 leu2delta0 ura3delta0 met15delta0

(Note: "STE2pr " represents the MATa-specific STE2 promoter; "Sp_his5 " represents the S. pombe his5 , which complements S. cerevisiae HIS3 ; also, this strain carries a wild-type LYS2 )

Other SGA starting strains carrying different reporters are available upon request.

Y8205

Starting strain for SGA query strain construction by switching.

Mating type: MAT alpha
Genotype: can1delta ::STE2pr-Sp_his5 lyp1delta ::STE3pr-LEU2 his3delta1 leu2delta0 ura3delta0

Upon request, we send out an SGA kit that contains: p4339, for switching kanMX deletion alleles to natMX alleles; Y7092, our standard SGA starting strain, into which query mutations are introduced by transformation; Y8205, another SGA starting strain, into which query mutations can be introduced by the switching protocol.

Hongweir with the robots.

Hongwei and Ermira in the robot room.

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Automation

Recently, we worked with two groups to develop robotic systems for manipulating high-density yeast arrays. The RoTor is a desktop system, designed for individual labs; it uses plastic pinning pads and makes manipulating yeast arrays very simple and rapid. The BioMatrix is for more of a power user; it is higher throughput, fully automated and can include stackers and cherry picking options.

RoTor HAD

BioMatrix

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This page was last updated on Monday, 28 May, 2012 7:10 PM