LiAc/SS-DNA/PEG TRANSFORMATION
Updated 4/15/2016 08:38 PM
Introduction
Materials
- YPD
- 1M LiAc
- 100mM LiAc
- 50% w/v PEG
- 10mg/mL SS-DNA
- SCM-minus plate
Procedure
- Inoculate 5mL of liquid YPD or 15mL SCM and incubate with shaking overnight at 30oC.
- Inoculate into 10mL YPD (5mL cells/transformation) to make OD600=0.1.
- Incubate the culture at 30oC on a shaker at 200 rpm until OD600~0.6-1.0. This will take around 6 hours.
- Harvest the culture in a sterile 50mL centrifuge tube at 3000 x g (5000 rpm) for 5 min. Keep cells cold from this step.
- Pour off the medium, resuspend the cells in 25mL of sterile water and centrifuge again.
- Pour off the water, resuspend the cells in 1.0mL 100mM LiAc and transfer the suspension to a 1.5mL microfuge tube.
- Pellet the cells at top speed for 15 sec and remove the LiAc with a micropipette.
- Resuspend the cells to a final volume of 500µL (2 x 109 cells/ml) --about 400µL of 100mM LiAc.
- Boil a 1.0mL sample of 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.
- Vortex the cell suspension and pipette 50µL samples into labelled microcentrifuge tubes. Pellet the cells and remove the LiAc with a micropipette.
- The basic "transformation mix" consists of:
240 µl PEG (50% w/v)
36µL 1.0M LiAc
10µL SS-DNA (10mg/mL)
X µl Plasmid DNA (0.1 - 10µg)
74-X µl Sterile ddH2O 360µL TOTAL
- Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min.
- Incubate at 30oC for 30 min.
- Heat shock in a water bath at 42oC for 30 min.
- Microfuge at 6-8000 rpm for 15 sec and remove the transformation mix with a micropipette.
- Pipette 200µL of sterile water into each tube and resuspend the pellet by pipetting it up and down gently.
- Plate transformation mix onto SCM-minus plates. For drug markers, plate on YPD plate and replica plating onto proper selection plates the next morning.
- Incubate the SCM minus plates for 2 - 4 days to recover transformants.
This protocol was created by Justin D Smith and edited by Hannah Shen. This is a standard lithium acetate protocol for transforming yeast with miniprep DNA. We use this protocol to get up to 22 million transformants/ µg of plasmid DNA.