High efficiency LiAc transformation

Tuesday, November 18, 2003

Description
High efficiency LiAc transformation

Procedure
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.

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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.

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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.

Recipes
10X LiAc 1 M LiAc, filter sterilize

50% PEG 3350, filter sterilize

Supplies

Tips

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High efficiency LiAc transformation