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High efficiency LiAc transformation

Modified from Gietz Homepage.
Stuff you need:

10X LiAc 1 M LiAc, filter sterilize

50% PEG 3350, filter sterilize



1. Inoculate 2-5 mls of liquid YPED or 10 ml synthetic media and incubate with shaking overnight at 30o C.

2. Count o/n culture and inoculate 50 mls of warm YPED to a cell density of 5 x 106/ml culture.


Note:

    i) Dilute overnight cultures 10-1 or more in water.

    ii) Carefully place 10 l of the cell suspension between the cover slip and the base of haemocytometer. Let the cells settle onto the haemocytometer grid for a few minutes. The grid area is typically 1 square millimeter, divided into 25 equal-sized squares, and the volume measured is 10-4 ml.

    ii) Count the number of cells in 5 diagonal squares

    iv) Calculate the cell titer as follows: cells counted x 5 x dilution factor x 1/volume measured by the 25 squares of the haemocytometer. 239 cells x 5 x 10 (dilution factor) x 1/10-4ml = 1.2 x 108 cells/ml.

    v) Saccharomyces cerevisiae divides by budding from a mother cell. Count budded cells as a single cells. Count cells with equal bud sizes as two cells there is evidence of additional buds forming on either cell.

    vi) You may also use OD660 to determine cell titer, however the relationship between cell number and OD is strain specific.


3. Incubate the culture at 30o C on a shaker at 200 rpm until its equivalent to 2 x 107 cells/ml. This will take 3 to 5 hours. This culture will give sufficient cells for 10 transformations.

Note:

    i) It is important to allow the cells to complete at least two divisions.
    ii) Transformation efficiency remains constant for 3 to 4 cell divisions.

4. Harvest the culture in a sterile 50 ml centrifuge tube at 1000 x g for 5 min.

5. Pour off the medium, resuspend the cells in 25 ml of sterile water and centrifuge again.

6. Pour off the water, resuspend the cells in 1.0 ml 100 mM LiAc and transfer the suspension to a 1.5 ml microfuge tube.

7. Pellet the cells at top speed for 15 sec and remove the LiAc with a micropipette.

8. Resuspend the cells to a final volume of 500 l (2 x 109 cells/ml) -- about 400 l of 100 mM LiAc--

Note:

    If the cell titer of the culture is greater than 2 x 107 cells/ml the volume of the LiAc should be increased to maintain the titer of this suspension at 2 x 109 cells/ml. If the titer of the culture is less than 2 x 107 cells/ml then decrease the amount of LiAc.

9. Boil SS-DNA for 5 min. and quickly chill in ice water.

    **** It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.****

10. Vortex the cell suspension and pipette 50 l samples into fuge tubes. Pellet the cells and remove the LiAc with a micropipette.

11. The basic "transformation mix" consists of:

    240 l PEG (50% w/v)
    36 l 1.0 M. LiAc
    50 l SS-DNA (2.0 mg/ml)
    X l DNA
    34-X l Sterile ddH2O
    360 l TOTAL

    Carefully add these ingredients in the order listed.

Note:

    The order is important here! The PEG should go in first, which shields the cells from the detrimental effects of the high concentration of LiAc.

    One can also premix the ingredients except for the DNA then add 355 l of TRAFO mix ontop of the cell pellet. Then add 5 l of DNA and mix. Take care to deliver the correct volume as the TRAFO mix is viscous.

12. Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min.

13. Heat shock in a water bath at 42o C for 40 min.

Note:

    The optimum time can vary for different yeast strains. Please test this if you need high efficiency from your transformations.

14. Microfuge at 6-8000 rpm for 15 sec and remove the transformation mix with a micropipette.

15. Pipette 600 l of sterile water into each tube and resuspend the pellet by pipetting it up and down gently.

Note:

    Be gentle as possible at this step if high efficiency is important.

16. Plate!

Note:

    When spreading yeast inoculum onto the plate gently distribute the fluid completely with a sterile glass rod with a minimum of strokes. Allow the fluid to be taken up by the plate prior to incubation.

17. Incubate plates for 2 - 4 days to recover transformants.


'Lazy Bones' transformation (plasmid transformations)

PLATE solution
40% PEG 3350
0.1M LiAc
10 mM Tris-HCl, pH 7.5
1 mM EDTA

  1. Take 0.5 ml of culture and spin 10 sec in microfuge. Decant the tube by inverting it and shaking it once. Alternatively, one can pick a colony (2-3 mm in diameter) from a plate with a toothpick and transfer cells to sterile 1.5 ml microfuge tube (as long as the plate is not dried out, colonies can be used from plates stored in the fridge for 3 months, maybe more).

  2. Add 10 l of carrier DNA (100 g) plus 1 g transforming DNA(in 10 l) and vortex well. (Carrier DNA does not need to be added if the transforming DNA has come from mini-prep DNA which has not been RNased).

  3. Add 0.5 ml PLATE solution and vortex.

  4. Add 57 l DMSO and vortex briefly.

  5. Leave for 15 min at RT.

  6. Heat shock for 15 min at 42 deg C.

  7. Pellet cells in microfuge for a few seconds at 10 krpm. Carefully remove supernatant.

  8. Add 200 l TE to the cell pellet and gently resuspend cells by aspirating up-and-down with a pipette tip. Immediately spread suspended cells onto selective plates.

    NOTES

    - the yield of transformants increases linearly up to about 100-200 g of transforming DNA.

    - the optimal number of cells per transformation is about 2E8 cells/ml. Cells + DNA volume should be about 140 l. In other words, PLATE:(Cells + DNA) should be about 3.5:1.


    PLATE Transformation

    Adapted from protocol used in the Rine lab.

    Necessary Solutions:
    1X TEL buffer
    10 mM Tris-HCl, pH 7.5
    1 mM EDTA
    0.1M LiAc

    PLATE solution
    40% PEG 3350
    0.1M LiAc
    10 mM Tris-HCl, pH 7.5
    1 mM EDTA

    COMPETENCY:
    1. Inoculate a single colony into 100 ml YPD (or selective media if necessary).

    2. Grow on shaker to O.D.(600) = 1.0 (O.D.(600) between 0.6 and 1.8 is fine) at 30oC overnight.

    3. Spin down cells in table top centrifuge at 2,000 rpm.

    4. Resuspend cells in 10 ml TEL.

    5. Shake vigorously overnight at room temperature; you can skip this step and proceed to the next if you don't want to save cells.

    6. Spin down cells and resuspend in 1 ml TEL.

    7. Use for transformation. Cells can be kept in the refrigerator and used for up to a month.

    TRANSFORMATION:
    1. Add 50 ug salmon sperm DNA (usually 5 ul of 10 mg/ml) to 100 ul of competent cells in a sterile 1.5 ml tube.

    2. Add DNA to suspension. Typically 1 ug of qiagen DNA, or 5 ul of miniprep DNA for uncut plasmids. Use more for integrating constructs.

    3. Incubate 30 minutes at room temperature.

    4. Add 0.7 ml PLATE solution, resuspend thoroughly.

    5. Incubate 1 hour at room temperature.

    6. Heat shock at 42oC for 5-10 minutes. Plate on appropriate selective plates.


    Electroporation

    1. Inoculate 50ml YEPD with a colony and grow with shaking at 30 deg C until early stationary (~0.6-2E8 cells/ml).

    2. Harvest in a 50ml sterile conical tube in GPR centrifuge spin at 3000rpm, 4 deg C, 5' and keep cells on ice throughout the procedure.

    3. Wash cells with 40ml ICE-COLD sterile dH2O, pellet at 2.5krpm, 4 deg C, 5'.

    4. Repeat wash with 20ml sterile dH2O (ice-cold).

    5. Resuspend cells in 5ml 1M Sorbitol (ice-cold) pellet at 2krpm, 4 deg C, 5'.

    6. Resuspend cells with 150l 1M Sorbitol (ice-cold) - KEEP ON ICE!

    7. Mix 40l of yeast suspension with <5l DNA (~5g) in a prechilled electroporation cuvette (0.2 cm). Tap contents to the bottom, making sure that the sample is in contact with both sides of the aluminum cuvette.

    8. Give one pulse: V= 1.5kV, 25F, 200 Ohms. Time should be ~4-5".

    9. Immediately add 1ml 1M Sorbitol (ice-cold) and transfer with a sterile pasteur pipette to a sterile Eppendorf tube.

    10. Spread onto selective plates.

 
Prepared By:
Michael McMurray
(206) 667-6660
Last Modified: Dec 6, 2002


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