Maitreya Dunham August
2004
Original protocol from
Katja Schwartz
Digest plasmid DNA so you
cut in a region of homology, leaving at least a couple hundred basepairs of
homology on both sides.
Gel purify it if desired.
Check concentration.
For a difficult
transformation, you will need ~10 µg DNA.
For an easier transformation, you can use less.
Maintain sterility
throughout. Whenever shaking is
called for, use the lowest setting that allows for complete mixing.
Inoculate 2 ml YPD with a
fresh colony.
Grow overnight 30¡C.
Inoculate 50 ml YPD with
200 µl of the overnight.
Monitor the growth until
the OD600 is around 0.7-0.8 (~7 hours).
Spin down the cells.
Resuspend in 5 ml lithium
acetate mix. Spin.
Resuspend in 0.5 ml
lithium acetate mix. Transfer to
an eppendorf tube.
Incubate 60 minutes at
room temperature on the orbital shaker.
Mix:
10 µl 10
mg/ml boiled sheared salmon sperm DNA
~10 µl ~5-10
µg DNA (also set up a ÐDNA control)
200 µl cells
Incubate 30 min at room
temperature on the orbital shaker.
Add 1 ml PEG mix.
Incubate 30 min at room
temperature on the orbital shaker.
Incubate 10 min 42¡C.
Spin down.
Remove ALL of the
supernatant with a pipet.
Resuspend the cells in 200
µl TE and plate to selective media.
For drug selections, you
may want to outgrow first.
Once transformants appear,
colony purify them.
Check the integration by
PCR with one flanking region primer and one internal primer, or with flanking
primers that give different size products.
Lithium acetate mix
10 ml 10X
TE
10 ml 1
M lithium acetate
80 ml water
Filter sterilize.
PEG mix
8 ml 50%
PEG 3500
1 ml 10X
TE
1 ml 1
M lithium acetate
Filter sterilize.
10X TE
10 ml 1
M Tris pH 7.5
2 ml 0.5
M EDTA pH 8
88 ml water
Filter sterilize.