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This is a quite simple but reliable protocol to make very high transformation efficiency yeast competent cells. By express your gene of interest, protein function can be studied in yeast cells.

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[Bio101] Making Yeast Competent Cells and Yeast Cell Transformation

Cell Biology > Cell isolation and culture > Transformation
Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
7/20/2011, 19441 views, 2 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.96

[Abstract] This is a quite simple but reliable protocol to make very high transformation efficiency yeast competent cells. By express your gene of interest, protein function can be studied in yeast cells.

Materials and Reagents

  1. Bacto-Yeast extract (Thermo Fisher Scientific)
  2. Bacto-peptone (Thermo Fisher Scientific)
  3. Glucose (dextrose) (Thermo Fisher Scientific)
  4. Bacto-agar (Thermo Fisher Scientific)
  5. Deionized H2O
  6. Glycerol (Sigma-Aldrich)
  7. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich)
  8. PEG 3350 (Sigma-Aldrich)
  9. Lithium acetate dihydrate (LiAc) (Sigma-Aldrich)
  10. Salmon sperm DNA (Life Technologies, Invitrogen™)
  11. Yeast nitrogen base (YNB) (Sigma-Aldrich)
  12. 2-(N-morpholino) ethanesulfonic acid (MES)
  13. ADE (Sigma-Aldrich)
  14. Agar
  15. YPAD plate & liquid medium (see Recipes)
  16. Transformation solution (see Recipes)
  17. YNB + MA plate (see Recipes)

Equipment

  1. Water bath (VWR International)
  2. Centrifuges (Eppendorf)
  3. 30 °C shaker and incubator (VWR International)
  4. Standard petri dishes (VWR International)
  5. 1.5 ml centrifuge tubes (Eppendorf)

Procedure

  1. Make yeast competent cells (Modified from Gietz & Schiestl, 2007)
    1. Obtain yeast strains of interest and streak on YPAD plates. Let cells grow 2 d before inoculation.
    2. 1st Inoculation: Inoculate one colony into 25 ml YPAD liquid medium. Grow cells overnight at 30 °C with shaking speed around 200 rpm.
    3. 2nd inoculation. Transfer the 25 ml cell culture into 75 ml YPAD medium. Grow cells at 30 °C for 4 h.
    4. Harvest cells by centrifugation at 3,000 x g for 5 min, wash cells with 0.5 volumes of sterile H2O.
    5. Centrifuge again with 3,000 x g for 5 min.
    6. Re-suspend cells in 0.01 volumes of sterile H2O, transfer to a suitable centrifuge tube and pellet at 3,000 x g for 5 min at 20 °C.
    7. Re-suspend cell pellet in 0.01 volumes of filter sterilized frozen competent cell solution (5% v/v glycerol, 10% v/v DMSO).
    8. Dispense 50 µl cells into 1.5 ml Eppendorf tubes.
    9. Place the tubes into a box with Styrofoam or cardboard (slow freezing is essential for good survival rates).
    10. Store the box in a -80 °C freezer (cells can be kept at this condition for up to one year).

  2. Yeast cell transformation
    1. If using competent cells stored at -80 °C, thaw cells at 37 °C water bath for 15-30 sec. if using freshly made competent cells, go to step 2).
    2. Centrifuge at 13,000 x g for 2 min to remove the supernatant.
    3. Make the transformation solution for the planned number of transformations plus one extra (negative control) (see the recipes).
    4. Add the solution to the cell pellet, vortex to re-suspend the cells.
    5. Incubate in a 42 °C water bath for 40 min.
    6. Centrifuge at 13,000 x g for 30 sec and remove the supernatant.
    7. Pipette 1 ml of sterile H2O into the transformation tube to re-suspend the pellet.
    8. Plate 200 µl of the cell suspension onto the YNB + MA plate growth plate with your selection marker.
    9. Incubate plates at 30 °C for 2~4 d.

Recipes

  1. Make YPAD plate & liquid medium
    1% Bacto-yeast extract - 10 g/L
    2% Bacto-peptone -20 g/L
    2% Glucose (Dextrose) - 20 g/L
    If making YPD plates, add 20 g Bacto-agar.
    Fill up to 1 L with deionized H2O.
    NO pH adjustments.
    Note: Autoclave agar and glucose separately, to avoid caramelization.
  2. Transformation solution
    PEG 3350 [50% (w/v)] 260 µl
    1 M LiAc 36 µl
    Salmon sperm DNA (10 mg/ml) 10 µl
    Plasmid 3 µl
    Sterile H2O 51 µl
    Total 360 µl
  3. YNB+ MA plate (100 ml)
    YNB 0.67 g
    20 mM MES 0.39 g
    ADE 0.01 g
    Agar 2 g
    Glucose 2 g

References

  1. Gietz, R. D. and Schiestl, R. H. (2007). Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 1-4.
  2. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M. A., Uozumi, N., Cheung, A. and Sze, H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93.


How to cite this protocol: Lu, Y. (2011). Making Yeast Competent Cells and Yeast Cell Transformation. Bio-protocol Bio101: e96. DOI: 10.21769/BioProtoc.96; Full Text



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5/17/2012 2:52:03 AM  

Casey
Roanoke College

In step 4, does the 0.5 volumes refer to the pellet size, the container to centrifuge, or the whole culture (100mL)?

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3/14/2012 7:23:19 PM  

How yeast cell is transformed?

3/21/2012 2:35:50 PM  

Yongxian Lu (Author)
Carnegie Institution for Science,Stanford University

The transformation is heat-shock based: treat cells with 42 degree loosens membrane and so DNA can get in.

Reply

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