Hello Debajyoti D,

          We are using Saccharomyces cerevisiae as a model system. This protocol is same for almost all laboratory yeast yeast. You did write but some important component you did not use like DMSO (makes cell porous) and Carrier DNA, both will increase competency and transformation efficiency.

Try once more this protocol

Here you go……

1xLiTE 0.1 M Lithium Acetate (1M stock)

1x TE (10 mM Tris-HCl pH7.9, 1 mM EDTA)

• 1.1X TE/LiAc Solution

Combine 1.1 ml of 10X TE

Buffer with 1.1 ml of 1 M LiAc (10X). Bring the total volume to 10 ml using sterile, deionized H2O.

• PEG/LiAc Solution (polyethylene glycol 3350/lithium acetate)

To prepare 10 ml of solution

8 ml of 50% PEG 3350

TE buffer 1X 1 ml of 10X TE Buffer

LiAC 1X 1 ml of 1 M LiAc (10X)

• 0.9% (w/v) NaCl Solution

0.9 g of NaCl in 100 ml of deionized H2O and filter-sterilize the solution

Yeast Transformation Protocol

A.    Protocol: Preparation of Competent Yeast Cells

1. Inoculate one colony 3 ml YPD medium in a sterile 15 ml culture tube.

2. Incubate at 30° C with shaking at 250 rpm for 12 hr.

3. Transfer 10 μl of the culture to 5 ml of YPD in a 50 ml folkan

6. Incubate shaking until the OD600 reaches 0.4–0.8 (16–20 hr).

7. pellet down 5 to 10 OD of cells (600A) by centrifuge 5000 rpm / 5 min

12. Discard the supernatant and wash pellet by resuspend in  600 μl of 1.1xTE/LiAc.

9. Discard the supernatant and resuspend pellet in  600 μl of 1.1xTE/LiAc The cells are now ready to be transformed with plasmid DNA, keep in ice.

B.     Protocol: Transformation of Competent Yeast Cells

1. Materials:

•Carrier DNA (single stranded DNA, salman sparm DNA,10 μg/μldenatured)

• Competent Yeast Cells(above protocal)

• PEG/LiAc solution(above)

• 0.9% (w/v) NaCl

• DMSO

Combine the following in a pre-chilled, sterile tube:

1.      Plasmid DNA (.500 ng to 1-2 μg/μl)

2. 5μl Carrier DNA (single stranded DNA, salman sparm DNA,10 μg/μl, denatured 90 oC for 10 min)

3. Add competent cells (50μl) and gently mix.

4. Add PEG/LiAc and gently mix.

5. Incubate at 30° C for 30 min.

6. Add DMSO (20μl/ tube) and gently mix x.

7. Place the tube in a 42° C water bath for 15 min.

8. keep in ice for 10 min

9. Centrifuge to pellet yeast cells, 5k/5min.

10. Remove the supernatant and resuspend in YPD Medium.

11. Incubate at 30° C for 1 h with shaking 200 rpm.

12. Centrifuge to pellet yeast cells, 5000 rpm/ 5 min

13. Discard the supernatant and resuspend in 100 – 200 μl, of  0.9% (w/v) NaCl Solution.

14. Spread on appropriate plate (-URA, -leu, -his or – trp…….. etc) depending on plasmid.

Thank you Praveen,

Thank you so much for the procedure!! I will follow it for S. cer then

you try both,

it will work

all the very best

Dear Debajyoti,

I have a few concerns about your transformation method since evrything seems OK but one little problem: where is denaturated sonicated salmon DNA added????????????

It seems you do not add denaturated sonicated salmon DNA and if so, you will get NO transformants at all (exactly what you observe) because these 50 micrograms of single-stranded small pieces of fish DNA binds to the very numerous nonspecific DNA-binding sites present at the surface of yeast cells and in the yeast wall !!! If you do not add it (as presumably you did) your cherished plasmidic DNA will be kept in these sites and will NEVER reach the cell inside !!

Best regards,

Pr Philippe Urban

Thanks,

But the protocol I used did not say anything for the salmon DNA. However, I will keep an option. Thanks for your suggestion Philippe.

Dear Debajyoti,

This has not to be specifically salmon DNA it can be DNA from other species. This consists in genomic DNA that was sonicated and which is boiled to 90°C for 10 min then directly put on ice. This way it yields a solution of pieces of single stranded DNA called "carrier DNA".

This is NOT an option in yeast transformation but a NECESSITY, otherwise transformation efficiency drops so abruptly that it does not work at all.

Best regards and good luck for yoru experiments,

Philippe Urban

Hi Debajyoti,

I have also experienced problems in the past with  S. pombe transformations, for me the problem was loss of cell viability after the LiAc (pH 4.9) incubation.  It might be good to plate out an aliquot of cells onto YE after each step in the transformation protocol to ensure that you have viable cells throughout the transformation procedure.

Are you transforming a plasmid or cassette for gene deletion? You could also try using the Bahler et al. protocol which uses LiAc at pH 7.5, DMSO and carrier DNA which improves transformation efficiency.  This protocol is for S. pombe gene deletions/tagging but has also been used for plasmid transformations:

http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0061(199807)14:10%3C943::AID-YEA292%3E3.0.CO;2-Y/abstract

Good luck!

Kerry

I am using Plasmid DNA. Yeas I was also suggested by others to follow the Bahler et al. protocol. Thanks for your input. I will keep note of cell viability in each steps.

I have tried all these protocols and they work really well. 

I particularly like the Freezing glycerol method (link provided below) as that allows you to store them in competent form. 

Good luck.

http://www.ncbi.nlm.nih.gov/pubmed/16088874

I used the method Aashiq H Kachroo recommended (the freezing glycerol method) and it worked really well. I grew the cells up in YES to prep the frozen cells instead of SLD liquid medium, and I used the EMM+leu dropout mix plates as selective plates instead of SLD plates. I got about 350 colonies. It was a very good method that doesn't take a lot of time.

Thank you Heng Liang