McClean:Blue Light Overview
Working with blue light constructs (our CRY2 and CIB1- based induction system) requires working knowledge in yeast genetics and plasmid construction, simple electronics, and programming (in the Arduino environment.)
Plasmid & Strain Considerations
Generally, our CIB1 constructs are in plasmid backbones that are LEU2+, our CRY2 constructs are in those that are TRP1+, and our reporter constructs are in those that are URA3+. Thus, if following this convention, you must be using a strain that is prototrophic for these (i.e. LEU2, TRP1, and URA3 deficient.) We generally use either one of two strains for these purposes, yMM1146 or yMM1204. Most of our blue light plasmids are constructed with the pRS41(x) vectors (Sikorski and Hieter, 1989.) These are in our collection as pMM5-8. Note that these are the CEN/ARS (read: single copy) plasmids.
Setting Up an LED Array for Testing
If you need to test your constructs to see if they "work," this is best performed in batch on a roller drum. Arrange three LED's (460 nm emittance for this experiment) at the 3, 9, and 12 o'clock positions of the roller drum so that they face the side of the drum (where they will be able to shine directly on culture tubes. Hook up the LED's as follows:
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.