Candida albicans Lithium Acetate DNA Transformation
Day1
1.) Using strains from the -80°C freezer, inoculate 2ml YPAD (+glucose) with the strain to be transformed. Incubate this culture overnight at 30°C while shaking/spinning.
2.) PCR 10- 50 ml reactions of transformation cassette and prepare cassette for transformation as indicated below. (For most cassettes, if the plasmid has been purified with the Qiagen kit, a final concentration of 2ng/ml is more than enough template for PCR. I use 10ml of plasmid at 50-100ng/ml for 10-50ml PCR reactions. I use a final concentration of 1mM of each primer.)
a. After PCR, remove 3 ml PCR product to run on a gel, then combine the rest of the PCR product in a 1.5 ml eppendorf tube and add 750 ml 95% Ethanol and 50 ml 3M NaOAc to precipitate. Vortex this tube and incubate at least 30 mins. at -20°C. (Longer is better.)
b. Spin down the precipitated cassette DNA at 16.1 (x 1,000) rcf for 10 mins. Discard supernatant in ethanol waste and dry DNA in the 37°C incubator (not shaking.) DNA is dry when the pellet becomes clear/white.
c. Resuspend dried cassette DNA in 40 ml TE pH 7.5 and set aside until step 7.
Day 2
3.) Innoculate 0.5 ml of the overnight yeast culture into 50 ml YPAD (+glucose) in a 125+ ml flask. Incubate the flask, while shaking, at 30°C for 3 hrs.
Note: If the strain you are transforming is sick or grows slowly you may need to grow cells for more than 3 hrs. Check to see if cells are ready to transform by looking on the microscope- most cells should have various-sized buds. If only large buds are present and the culture is thin, cells may need to be grown longer.
4.) Pour cultured cells into a 50 ml centrifuge tube and pellet them by spinning at 1,500 rpm for 5 minutes.
5.) Discard the supernatant and resuspend cells in 10 ml ddH2O, then pellet the cells as before.
6.) Discard supernatant and resuspend the washed pellet in 0.5 ml TELiOAc. Set this aside until step 9.
7.) Add 5 ml sheared salmon sperm (carrier) DNA at 10 mg/ml to each of two eppendorf tubes.
8.) Add 40 ml of resuspended transformation cassette (from step 2) to one of the tubes with carrier DNA and nothing to the other tube (this is the negative control.)
9.) Add 0.1 ml aliquots of the cell suspension in TELiOAc to each tube with carrier DNA and mix by pipetting up and down. Incubate tubes at room temperature for 30 minutes.
10.) Add 0.7 ml of plate mix (see recipe below) to each tube and mix by pipetting up and down.
11.) Incubate at room temperature overnight.
Day 3
12.) Heat shock the cells at 42°C for 1hr.
13.) Pellet the cells in a centrifuge at 8 (x1000) rcf for 30 seconds (be gentle with them since they are fragile at this point.)
14.) Remove the supernatant with a micropipette and resuspend the cells in 0.1 ml ddH2O.
15.) Pipette cells onto appropriate selective (SDC-) plates and spread by adding ~15 large glass beads and swirling. (Use one plate for the control and a second for the transformation.)
16.) Place plated cells at 30°C for 3+ days. Hope that something grows (but not the first day or on the control plate because this indicates contamination.)
Plate Mix Recipe:
10 mM Tris HCl, pH 7.5 / 8.0
1 mM EDTA, pH 8.0
0.1 M Lithium Acetate
40% PEG 3350
For 10 ml Plate Mix: 20ml 50ml
100 µl 1M Tris 200 µl 500 µl
20 µl 0.5M EDTA 40 µl 100 µl
1 ml 1M LiOAc 2 ml 5 ml
8 ml 50% PEG 3350 16 ml 40 ml
700 µl ddH2O 1.4 ml 3.5 ml
TELiOAc 10 ml:
100 µl 1M Tris
20 µl 0.5M EDTA
1 ml 1M LiOAc
8.7 ml ddH2O
50% PEG 100 ml:
Weigh 50g of PEG 3350 (powder) and pour this into a 150+ ml beaker or flask. Slowly add ddH2O while mixing until the volume reaches 100 ml. Place a stir bar in the beaker to spin gently overnight. In the morning, it may be necessary to add additional ddH2O to bring the volume back up to 100 ml again. Pour the mixture in a bottle and autoclave.