Expt. 3 Cloning by complementation  (2 weeks total)

In this experiment, we will clone a yeast gene by complementation.  We will start with a yeast strain that contains a temperature sensitive mutation (ebp2-1)in an as yet minimally characterized essential gene (EBP2).  Cells harboring this ebp2-1 mutation are unable to grow at the restrictive temperature of 36°C.  We will take an ebp2-1 strain and transform it with a plasmid library containing random inserts of yeast genomic DNA.  We will then test these transformants to see which colonies can now grow at the restrictive temperature of 36°C (complementation).  Only those cells that gain a piece of yeast genomic DNA that can complement the ebp2-1 allele should grow at 36°C.  This genomic DNA could contain either the authentic EBP2 gene, or possibly a suppressor of the ebp2-1 mutation.  To investigate these possibilities, we will isolate the plasmids from the transformants, grow them in E. coli and analyze them by restriction mapping.

Strain to use

YMM55  Mat a  ebp2::HIS3  ura3-52  leu2  lys2  trp1  his3   (pMM112 ebp2-1  LEU2)
YMM179  Mat a ebp2-1.Kan ura3-52 leu2 trp1

Plasmids to use

YCP50, a URA3 shuttle vector

pMM113, a URA3 EBP2 shuttle vector

Genomic library, YCP50 containing random inserts of yeast genomic DNA

Day 1 Take an overnight culture of strain YMM and prepare for transformation as described in the separate protocal. At the end of the transformation, each sample of cells will be divided into two and plated onto 2 selective plates (SC-URA). One set will be incubated at 29 oC and the other will be incubated at 36 oC.

Tube DNA sample Incubation temp
1 no DNA 29 oC and 36 oC
2 1 ul YCP50 (URA3) 29 oC and 36 oC
3 1 ul pMM113 (URA3, EBP2) 29 oC and 36 oC
4 Plasmid Library pool A or B (URA3, genomic DNA) 29 oC and 36 oC


In an experiment like this, it is vital that you keep track of where your colonies came from. To keep track of your plates, label them as follows:

1-29 = Transformation sample #1 that was incubated at 29 oC

1-36 = Transformation sample #1 that was incubated at 36 oC
The transformants that you pick from these plates can then be labeled by letters


Day 4 or 5 Count and record the number of transformants on each plate.  Take 2 transformants each from plates 2, 3, and 4 and innoculate into the wells of a frog pond containing sterile water.  Also, innoculate one well with YMM55.  To the remaining wells, innoculate transformants from plates 5 and 6.  Be sure to keep a good record of which colonies went into which well.  Frog the cultures to two YPD plates, two 5-FOA plates, one SC and one SC - Ura plate. Incubate all plates at 25°C except the YPD and 5-FOA duplicates which should go in the 36°C incubator.

Day 7 Score the growth on the plates.  Take the following colonies from the SC - Ura  plate to innoculate into 3 mls of YPD and grow overnight on a rollerdrum at 25°C.  Take a colony that came from plate #2 (vector alone), #4 (positive control) and 3 colonies that came from tubes #5 or 6 that grew at 36°C on the YPD plate, but did not grow at 36°C on the 5-FOA plate.  These colonies have a URA3 plasmid that can complement the ts- ebp2-1 allele.

Day 8 Take the 5 overnight yeast cultures and do a "smash and grab" to recover the plasmids from the yeast strains into E. coli.  Use plasmid pMM112 as a positive control for the transformation and no DNA as a negative control.  After transformation, plate onto LB + Amp plates and incubate overnight at 37°C.
Tube DNA from No. of transformants
1 No DNA #
2 Plasmid prep pMM113 #
3 yeast culture 4-1 (pMM113) #
4 yeast culture 5-1 (library plasmid) #
5 yeast culture 5-2 (library plasmid) #
6 yeast culture 6-2 (library plasmid) #

Day 9 Score LB + Amp plates and put them at 4°C until day 14.

Day 14  If possible, innoculate 3 E. coli transformants that came from original tubes #5 or 6, plus 1 positive control (pMM113 transformant) into 3 mls of LB + Amp and grow O/N (over night) at 37°C (roller drum in warm room).

Day 15 Do a miniprep (see protocal) on the 4 E. coli cultures and  resuspend the DNA in 50 ul of T.E. buffer.  Take 10 ul of the DNA from each prep and digest with the restriction enzyme NdeI for 2 hours at 37°C.  Run cut and uncut samples on a gel and take a picture of the digests.

  10 ul  DNA in T.E.         T.E. is 10 mM Tris pH 7.5
      2 ul  10 X Buffer                         1 mM EDTA
      7 ul   water
      1 ul   Enzyme
     20 ul

Yeast Plasmid DNA prep
This protocol is used for recovering plasmids from yeast cultures.

 breaking buffer: 2% Triton X-100 (Sigma, X100)
    % SDS (BioRad, 161-030)
    100 mM NaCl
    10 mM Tris-Cl, pH 8.0
    1 mM EDTA, pH 8.0
 - T.E. buffer (pH 8.0): 10 mM Tris-Cl, pH 8.0
     1 mM EDTA, pH 8.0
 - chilled phenol:choloroform:isoamyl alcohol (25:24:1)
 - chilled 95% ethanol
  - acid-washed glass beads (Sigma, G 3753, See CPMB, 13.12.1)
- grow up yeast culture to appropriate density (near saturation)
- spin 1.5 mls of culture for 1 min in microfuge and aspirate off supernatant
- resuspend pellet in 200 ul breaking buffer
- wear gloves and add:
  200 ml phenol:choloroform:isoamyl alcohol (25:24:1)
  200 ml  (@200 mg) glass beads
  close cap tightly and vortex for  2.5 min.
Be careful when vortexing; label can be dissolved by the phenol.
 Hold cap tightly so it doesnât open or spill.
 - add 200 ml TE buffer and spin for 5 min, in microfuge
 - transfer 350  ml aqueous (top) layer to fresh eppendorf.
 - add 1 ml 95% ethanol  and mix well, let sit for 10 minutes
 - spin for 2 min, take off supernatant, and let dry upside down 10 min.
 - resuspend pellet in 50 ml TE buffer.

You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli .  Having the plasmid in E. coli  rather than yeast will make subsequent analysis easier.