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Yeast Protocols Handbook
V. Yeast Transformation Procedures continued
can be used either as an autonomously replicating plasmid or as an integrated plasmid, depending
on the desired level of reporter gene expression. The primary reason for integrating a plasmid in
some MATCHMAKER applications is to generate a stable yeast reporter strain in which only one
copy of the reporter gene is present per cell, and thereby control the level of background
expression. If you have an application that requires integration of a plasmid into the yeast genome,
please see Section V.D.
Transformation controls
When setting up any type of transformation experiment, be sure to include proper controls for
transformation efficiencies. In the case of simultaneous cotransformation, it is important to
determine the transformation efficiencies of both plasmids together, as well as of each type of
plasmid independently. That way, if the cotransformation efficiency is low, you may be able to
determine whether one of the plasmid types was responsible (see Troubleshooting Guide, Section F).
Therefore, be sure to plate an aliquot of the transformation mixture on the appropriate SD media
that will select for only one type of plasmid. Example calculations are shown in Section V.E. When
screening a library or performing a one- or two-hybrid assay, you will need additional controls, as
explained your system-specific User Manual.
B. Reagents and Materials Required
Note: The YEASTMAKER Yeast Transformation System (#K1606-1) contains all the solutions (except media, H2O, and
DMSO) required for yeast transformation. YEASTMAKER reagents have been optimized for use in the MATCHMAKER
One- and Two-Hybrid Systems.
• YPD or the appropriate SD liquid medium
• Sterile 1X TE/1X LiAc (Prepare immediately prior to use from 10X stocks; stock recipes in
Appendix D.B)
• Sterile 1.5-ml microcentrifuge tubes for the transformation
• Appropriate SD agar plates (100-mm diameter)
Notes:
• Prepare the selection media and pour the required number of agar plates in advance. (See your system-specific
User Manual or Appendix E for media recommendations.) Be sure to plan for enough plates for the control
transformations and platings.
• Allow SD agar plates to dry (unsleeved) at room temperature for 2–3 days or at 30°C for 3 hr prior to plating any
transformation mixtures. Excess moisture on the agar surface can lead to inaccurate results due to uneven
spreading of cells or localized variations in additive concentrations.
• Appropriate plasmid DNA in solution (check amounts required)
• Appropriate yeast reporter strain for making competent cells (check volume of competent cells
required; Steps 1–11 of Section V.E will give you 1.5 ml, enough for 14–15 small-scale
transformations)
• Herring testes carrier DNA (Appendix D.B)
• Sterile PEG/LiAc solution (Prepare only the volume needed, immediately prior to use, from
10X stocks; Appendix D.B)
• 100% DMSO (Dimethyl sulfoxide; Sigma #D-8779)
• Sterile 1X TE buffer (Prepare from 10X TE buffer; Appendix D.B)
• Sterile glass rod, bent Pasteur pipette, or 5-mm glass beads for spreading cells on plates.
Protocol # PT3024-1
Version # PR13103
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