Transformation of Filamentous Fungi.
1. M. grisea: cut a 1" square of mycelium from an CM plate and blend in 50 ml
complete medium. Incubate at 25oC, with shaking for 48 h
2. Harvest the culture by filtration through sterile Miracloth and wash mycelia in
sterile water.
3. Transfer mycelia to a 50 ml conical tube. Add 40 ml OM buffer/500 mg Glucanex
(pH 5.5 – 5.8 filter sterilised ) and shake gently to disperse hyphal clumps. Incubate at
30oC with gentle (75 rpm) shaking, for 2 to 3 hrs.
4. Transfer protoplasts to sterile polycarbonate or polysulfonate Oakridge tubes and
overlay with an equal volume cold ST buffer. Spin at 5000 rpm/4oC in a swinging
bucket rotor. Protoplasts are recovered from the OM/ST interface. (At this point, until
the transformation, I keep the protoplasts and buffers cold. I don't know if it matters)
5. Transfer protoplasts to two or three sterile Oakridge tubes, and fill the tubes with
STC. This will adequately dilute out the OM and ST. Pellet protoplasts at 3000
rpm/10 min/4oC, in swinging bucket rotor. Wash twice more with approx. 10 ml STC.
Completely resuspend the pellet each time.
6. Resuspend protoplasts in 1.0 ml of STC and count microscopically. Yield should
be in the range of 2-5 X 108.
7. In a 1.5 ml eppendorf tube combine protoplasts (1 X 106 to 1.0 X 107; I routinely
use 5 X 106) with DNA (approx. 2 ug) in a total volume of 150 µl of STC. Incubate at
room temperature for 15 to 25 min.
8. Add 1.0 ml of PTC (in 2 to 3 aliquots), mix gently by inversion and incubate at
room temp. for 15 to 20 min.
9 Add protoplasts to 125-150 ml molten (45oC) agar medium (complete medium/0.8
M sucrose/1.5% agar), mix gently and pour into 5-6 plates (approx 20 -25 ml/plate).
Incubate for at least 16 hrs at room temp. and overlay with approx. 15 ml top agar
(minimal medium/1% agar) containing 300 ug/ml Hygromycin B.
notes:
1. Hygromycin B stock solution is 100 mg/ml in sterile ddH20
2. Select transformants and plate onto CM containing 100ug/ml Hygromycin B.
Sterile Ware
blenders
polycarbonate or polysulfonate Oakridge tubes
Miracloth
Plastic Funnels
pipette tips
eppendorf tubes
Buffers and Media
OM Buffer
1.2 M MgSO4
10 mM Na-PO4 (pH 5.8) diluted from a 1.0 M stock of
equimolar of mono and dibasic
Filter sterilize
ST Buffer
0.6 M Sorbitol
54.66 g
(500 ml)
0.1 M Tris-HCl pH 7.0
Autoclave
50 ml 1 M
STC Buffer
(500 ml)
1.2 M Sorbitol
10 mM Tris-HCl pH 7.5
10 mM CaCl2
109.32 g
5 ml 1 M
Autoclave
PTC Buffer
60% PEG 4000
60 g
(100 ml)
10 mM Tris-HCl pH 7.5
10 mM CaCl2
1 ml 1 M
1 ml 1 M
or
40% PEG 4000
1 M sorbitol
10 mM Tris-HCl pH 7.5
10 mM CaCl2
40 g
18.22 g
1 ml 1 M
1 ml 1 M
Autoclave