2 Hybrid System TRAFO Protocol



Note: Please cite

Agatep, R., R.D. Kirkpatrick, D.L. Parchaliuk, R.A. Woods, and R.D. Gietz (1998) Transformation of Saccharomyces cerevisiae by the lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ss-DNA/PEG) protocol. Technical Tips Online (http://tto.trends.com).

For complete instructions on how to do a two hybrid screen see the following references.

1. Gietz, R.D., B. Triggs-Raine, A. Robbins, K.C. Graham, and R.A. Woods (1997) Identification of proteins that interact with a protein of interest: Applications of the yeast two-hybrid system. Mol Cell Biochem 172:67-79.

2. Parchaliuk, D.L., R.D. Kirkpatrick, R. Agatep, S.L. Simon and R.D. Gietz (1999) Yeast two-hybrid system screening. Technical Tips Online (http://tto.trends.com) (accepted).not on line yet



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This TRAFO Protocol is for 2 Hybrid system screens
(We have done over 30 and still counting)



High Efficiency Transformation of a yeast strain requiring maintenance of a plasmid.



All solutions used in this protocol are described in the TRAFO Solutions Page


  1. Inoculate the yeast strain containing the first plasmid into the appropriate volume of the appropriate SC-omission medium in a flask and incubate at 30 oC overnight.

      For each different Scale up use the appropriate size of culture
       
      TRAFO SCALE 10 X 30 X 60 X
      Culture Size 25 mls 50mls 100 mls

       

  2. Determine the cell titer and calculate the volume of cells that yields 2.5 x 108 cells for each 50 mls of YPAD culture needed


       
      TRAFO SCALE 10 X 30 X 60 X
      YPAD culture Size 50 mls 150 mls 300 mls
      # of Cells needed 2.5 x 108 7.5 x 108 1.5 x 109

       

  3. Pour this culture volume into an appropriate sterile centrifuge tube and pellet the cells at 3000 x g for 5 min.
    Resuspend the cell pellet in the appropriate volume of pre-warmed (30o C) YPAD and transfer to another sterile culture flask.


       
      TRAFO SCALE 10 X 30 X 60 X
      YPAD culture Size 50 mls 150 mls 300 mls

       

  4. Incubate at 30o C while shaking at 200 rpm for 3 to 4 hrs until the cell titer reaches 2 x 107 cells/ml.
  5. Harvest the cells by centrifugation at 3000 x g for 5 min.
  6. Wash the cell pellet via resuspension with 1/2 volume of sdd water and collection by centrifugation as above.
  7. Resuspend the pellet in the appropriate volume of 100 mM sterile LiAc and transfer to an appropriate centrifuge tube. Incubate for 15 min at 30oC. Pellet the cells again by centrifugation and remove the supernatant.


       

      TRAFO SCALE 10 X 30 X 60 X
      100 mM LiAc 3 mls 3 mls 6 mls

       
  8. Add, in the order from top to bottom, the components of the transformation mix listed in the table below to a seperate tube and mix thoroughly by vortexing. Add the transformation mix to the cell pellet and vortex vigorously to resuspend the cell pellet. Alternatively you may also mix all components but the plasmid DNA together. Add the entire volume of the transformation mix minus the plasmid DNA to the cell pellet and then add the plasmid DNA and mix. This will keep you from loosing any plasmid DNA when transfering the viscous liquid of the transformation mix ontop of the cells.


       
      TRAFO SCALE 10 X 30 X 60 X
      50% PEG 2.4 ml 7.20 ml 14.40 ml
      1.0 M LiAc 360 l 1.08 ml 2.16 ml
      SS-DNA (2 mg/ml) 500 l 1.50 ml 3.00 ml
      Library plasmid DNA A l B l C l
      sdd Water 340 - A l 1.02 - B ml 2.04 - C ml

       


    Please note:
    The values for each scale up should be multiplied from the single reaction volumes. Previously the 60X scale up values for the LiAc, SS-DNA, and Plasmid DNA were NOT correct! (They were 90X scale, sorry) The numbers shown here are NOW correct! Thanks to the person that caught my error and sorry to all of you battling to get good 2HS screens done. In addition, Please note that we are now adding 2X the amount of carrier than in previous versions of this this page.

    TIP:

  9. Vigourously vortex the cell pellet until it is totally resuspended, which should take about 1 min. If you have problems getting the pellet resusupended let it sit for 5 min and then vortex!
  10. Incubate the transformation mix at 30o C for 30 min.
  11. Heat shock at 42o C for time indicated by table below with mixing by inversion for 15 sec after every 5 min.


       
      TRAFO SCALE 10 X 30 X 60 X
      Heat shock Time 30 min 40 min 45-60 min

       

    TIP:

  12. Collect the cells by centrifugation as above. Gently resuspend the cell pellet in an appropriate volume of sdd water and plate onto SC ommission medium. For our 30 X and 60 X 2 hybrid screens we plate onto 100 large plates. (Yes! that is correct! A whole case of large plates) This gives better transformation and library coverage! For the 10X screens we used less.


       
      TRAFO SCALE 10 X 30 X 60 X
      Resuspension Volume 10 mls 40 mls 40 mls

       

    TIP:

      Transformations for the two-hybrid system which use the activation of the HIS3 gene for genetic selection can be plated directly onto SC omission medium lacking Tryptophan, Leucine, and Histidine (Trp, Leu, His). The total number of transformants screened should be calculated by plating of a small aliquot (1- 2 l) onto a pair of SC omission medium lacking Trp-Leu plates.


  13. Incubate the plates for 3 - 5 days at 30o C or until colonies appear. For some two hybrid Screen we wait as long as 14 days!




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