Electroporation of Tetrahymena
1. Grow 100 ml of CU428 (VII) and SB1969 (II) strains in 2%PPYS +1X PSF,
shaking at 30°C, to a density of 3x105 cells/mL.
2. Spin cells at 3000 rpms for 3 minutes to pellet cells (quickly pour of supernatant
before cells swim up). Then add 50 mL of 10 mM Tris (pH 7.4) and spin again
to pellet.
3. Pour off supernatant and resuspend each strain in 75 mL 10 mM Tris (pH 7.4) and
put in an incubator (no shaking) at 30°C for 12-20 hrs. (ideally I use 18 hours and
it works best for me).
4. After about 5-6 hrs. Count the cells and adjust each strain culture to 3x105
cells/mL with prewarmed 10 mM Tris (pH 7.4) and place back in the incubator
until they have starved for 12-20 hrs total.
5. Alternatively, if your incubator has a timer on it you can mix the two strains in
equal proportions at this time and put them in a three 2 L flasks (making sure that
the volume of cells just covers the bottom of the flask (only have it a 3-5 mm
thick) and then setup the timer so that the shaking component turns off at 18 hrs.
6. If you starve them individually for 18 hrs., mix equal volumes of cells and place
in 11x13 glass dish (sterile) and place in incubator at 30°C. FROM THIS POINT
DO NOT TOUCH CELLS FOR 3-4 HRS. MIXING CAN PREVENT PROPER
PAIRING EFFICIENCY.
7. 4 hrs after mixing a sample of the cells should be taken and pairing efficiency
determined (if not greater than 75% do not continue) and then dry cells on slide
and DAPI stain to determine if most of the cell are in the same stage of
conjugation (~80% of pairs should be in meiotic prophase or later at 4 hrs.).
8. 9 hrs. after mixing a sample should be taken and DAPI stained quickly to
determine the percentage of cells in anlagen (two new MACs and two new MICs
with an old parental MAC). If there is 20% in anlagen wait 60 minutes then
continue to preparation for transformation. If 50% only wait 30 minutes. If less
than 20% check cells again in 60 minutes to determine percentage in anlagen.
NOTE: MY EXPERIENCE HAS SHOWN THAT SOME STRAINS TAKE UP
TO 11 HRS TO GET TO 75% ANLAGEN.
9. When cells are at 75% anlagen (9-11 hrs.). Spin down 100 mL cells at 3000 rpms
for 3 minutes and wash with 50 mL 10 mM HEPES (pH 7.4) and spin again.
10. Resuspend cells in 1 mL 10 mM HEPES (pH 7.4).
11. Take 125 µL of prepared cells and mix with 125 µL of plasmid DNA (rDNA
circular vectors ~30 µg DNA; linearized constructs for knockouts or integration ~
50 µg [See preparation of DNA for electroporation below]; adding more DNA
can help if you are getting very low transformation efficiency).
12. Place cells+DNA in electroporation cuvette (2 mm Gap) and electroporate (in
BTX 630 model: voltage = 230 V (LV); resistance = 25 omhs; capacitance = 175
µFarads). The peak voltage when cells are electroporated should be 221V with a
time constant of 3-4 msec.
13. Cells are let to recover for 1 minute and then placed into 25 mL of fresh 2%PPYS
+1xPSF in a 250 mL flask and placed at 30°C (no shaking) for 16 hrs.
14. Cells are then diluted with 25 mLs of 2%PPYS+1xPSF+ 180 µg/mL
Paromomycin (gives a final concentration of 90 µg/mL PM).
15. Cells are plated 500 µL per well in 2 48-well plates and placed in a humidity
chamber (to prevent evaporation) and placed at 30°C for 4 days.
16. On the 4th day after drugging with PM check the wells (If cells are still growing in
your NO DNA control add 500 µL of 300 µg/mL PM to each well to increase the
drug to ~200 µg/mL and put back in the incubator at 30°C overnight.
17. The next day score the number of positive wells and transfer to fresh 200 µg/ml
PM media (transfer 25µL into 500 µL of fresh media).
18. Positive cells will grow and should be further selected in PM to phenotypically
assort MAC to 100% (increasing drug to 1-5 mg/mL is usually good).
19. Confirm transformants by PCR, RT-PCR, or southern blot.
The preparation of the DNA for Electroporation:
rDNA vectors: prep DNA by lysozyme boil prep and RNAase treat (best to then
Phenol:Chloroform extract and EtOH precipitate to remove the RNase).
Knockout or btu1-1 knock-in:
1. The DNA (plasmid) is digested overnight to linearize it for better efficiency of
transformation (usually with SacI and KpnI) using around 120µg of DNA in a 200
µl reaction and using 5µl of each enzyme (with SacI and KpnI BSA is also
added). The reaction is then incubated overnight at 37C.
2. The next day the Digest is phenol:Chloroform, Chloroform, and ethanol
precipitated for 2 hrs-overnight. The precipitation is then centrifuged at max
speed for 10-20 minutes. The precipitate is washed with 70% ETOH and
centrifuged for 5 minutes and the ethanol removed.
3. The pellet is then dried to remove any ethanol and rehydrated in 20 µl of TE and
then the Digested DNA is Quatified and the concentration is adjusted to 1.0 µg/µl.