2 Hybrid System TRAFO Protocol

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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

SEE TRAFO REFs

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.

The two-hybrid system and other similar genetic screens in yeast involve the use of two different plasmids in a single yeast cell. One plasmid often contains a cloned gene or DNA sequence while the other plasmid contains a library of genomic or cDNA. While both plasmids can be co-transformed into a single yeast cell, it is often more efficient to transform the library pool into a strain already containing the first plasmid (R.D. Gietz, unpublished data).

To prepare a plasmid-carrying strain for an additional transformation, the initial growth phase should be using SC omission media until the cell titer reaches 1-2 x 10⁷ cells/ml. This will maintain the first plasmid during this growth phase. The cells can then be subcultured in YPAD medium for two doublings without significant plasmid loss, if the plasmid has no effect on yeast growth. To produce highly competent yeast cells containing a selectable plasmid, follow the protocol below. To determine the Scale up needed for the desired number of transformants, we recommend testing one's library DNA with the protocol below to determine the number of Transformants that can be expected. Then select the conditions that give a "good" Transformation yield and then scale up to 30X, 60X or even 120X obtain the desired number of transformants.( see Table 1 & Discussion).

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 (30^o 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 30^o 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 30^oC. 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 µl	1.08 ml	2.16 ml
   SS-DNA (2 mg/ml)	500 µl	1.50 ml	3.00 ml
   Library plasmid DNA	A µl	B µl	C µl
   sdd Water	340 - A µl	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:

   i. The standard transformation reaction can be scaled up to 120 X a standard transformation reaction, however we rarely need to go to this scale.

   ii. Use a a plastic pipet rather than a glass pipet to transfer the PEG solution as it adheres to the surface of glass pipets and hampers the delivery of an exact volume.

   iii. The volume of sdd water and plasmid DNA may be adjusted, however, the total volume of these components must remain constant.
   
   

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 30^o C for 30 min.
11. Heat shock at 42^o 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:

    Heat shock of large scale transformations require the culture tube to be inverted several times every 5 minutes to equilibrate the temperature quickly in the larger volume.
    

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 µl) onto a pair of SC omission medium lacking Trp-Leu plates.

    
    

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

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Discussion

It is important to optimize the amount of library plasmid DNA for each standard transformation. As shown in Table 1 below, such a test is done by transforming increasing amounts of library plasmid DNA in a standard transformation reaction. From this experiment, one can see that it is more productive to do 10 standard transformation reactions with 1 µg or plasmid DNA (or scale up the transformation reaction) than to do one standard transformation using 10 µg of plasmid. This will not only ensure efficient use library DNA but will also reduces the number of yeast colonies containing more than one library plasmid, which can make subsequent analysis difficult.

Co-transformation of library plasmid DNA with a GAL4_BD plasmid is not as efficient as transformation of the library DNA into a yeast strain already containing the GAL4_BD plasmid. Therefore, it is recommended that the GAL4_BD plasmid be transformed first into the yeast strain using the Quick & Easy protocol and followed by transformation of the library with protocol above; we have screened as many as 5.2 x 10⁷ transformants from a single scaled up transformation reaction in a two-hybrid screen (R.D. Gietz, unpublished data). In cases were the GAL4_BD fusion plasmid affects the growth of the yeast strain, it may be advisable to co-transform the GAL4_BD fusion plasmid and the library plasmid DNA. In these cases, careful attention to the levels of each plasmid in the transformation reaction is necessary for the production of the highest efficiency. BACK UP

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