Yeast Library Transformations
1. Grow the recipient strain of yeast to mid-log (1x107cells/ml). The better the media used the better the transformation efficiency, so use YPD when possible.
2. Pellet the cells and resuspend the pellets in LiTE (100mM LiAc in TE), the volume is not critical because this is a wash step.
3. Pellet the cells once more and this time resuspend the cells in 5ml of LiSORB for every 200ml of culture (LiSORB is LiTE containing 1M Sorbitol).
4. Incubate the cells for 15-30 minutes at 30°C with shaking. (Try not to go too long, cells begin to strarve and it decreases their survival)
5. Pellet the cells as above and resuspend in 500µl of LiSORB per 200ml of culture.
(I usually do large scale transformations with a liter of cells so the final volume is 2.5 mls)
6. After removing 100µl of cells for a negative control, add 2µg of transforming DNA and 200µg of Yeast total RNA for every 100µl of cells remaining. (50 ug DNA and 5 mg of total RNA carrier for 1 liters worth of cells)
7. Mix well then incubate for 10′ at 30°C without shaking.
8. Add 900µl of LiPEG (LiTE containing 40% PEG 3350) for each 100µl of cells and mix well. This is 22.5 ml for the large scale. I usually do this in 125 ml or 250 ml flasks. This aids the heat shock step later.
9. Heat shock the tubes or flask the flask in a 42°C water bath for 12 minutes.
10. At this point cells can be plated out to check the transformation frequency. 5 ul on a plate should give 1000 or more transformants with a good transforming strain. However, in order to plate a large number of cells per plate, we generally take our transformation mixture and add it to 500 mls of dropout minimal media for an initial 1 liter culture and allow it to recover at 30°C for 4 hours. At this point cells have been established as transformants. They can be pelleted, concentrated, and frozen at -70°C in 9% DMSO and stored for future use. Storing small aliquots allows you to thaw them and plate them out later at an appropriate density for whatever purpose you might desire in the future. For activation domain transformations we recover in leu, trp, his triple dropout media. It is important not to have extra his around during plating since it interferes with the 3-AT selection. We have been successful plating directly from recovered or from frozen cells.
Our experience has taught us not try to pellet the cells through the PEG since this lowers the transformation frequency, and not to wait too long to plate or grow up the transformants since the PEG is toxic to the cells. However, everything will work to some degree so try any variation on this protocol that might save you time.
1. Grow the recipient strain of yeast to mid-log (1×107). The better the media used the better the transformation efficiency, so use YPD when possible.
2. Pellet the cells at 2000 rpm in 50 ml aliquots in the Sorvall bench top centrifuge (using 50 ml Falcon® tubes).
3. Resuspend the cells in sterile water; the volume of water used is not important since your only washing the cells. (At this point I usually pooled the cells in 2 Falcon® tubes).
4. Repellet the cells as above and then resuspend the pellets in LiTE (100mM LiAc in TE); again, the volume is not critical.
5. Pellet the cells once more and this time resuspend the cells in 5ml of LiSORB for every 200ml of culture (LiSORB is LiTE containing 1M Sorbitol).
6. Incubate the cells for 1h at 30°C with shaking. (Try not to go too long, 1h is optimal 1.5h is dangerous)
7. Pellet the cells as above and decant.; resuspend in 250 µl of Carrier Resuspen-sion Buffer per 200ml of culture (CRB is prepared by diluting 20 mg/ml carrier DNA in LiSORB to a concentration of 4 mg/ml.)
8. After removing 100µl of cells for a negative control, add 2µg of transforming DNA for every 100µl of cells remaining.
9. Mix well, then incubate for 30′ at 30°C without shaking.
10. Add 900µl of LiPEG (LiTE containing 40% PEG 3350) for each 100µl of cells and mix well.
11. Divide the cell suspension in Eppendorf™ tubes (˜1ml per tube); this is important because it takes too long a time to heat a large volume of PEG uniformly.
12. Incubate the tubes at 30°C for 20 minutes.
13. Mix the contents of each tube and then heat the tubes (in a heating block) for 10′ at 42°C.
14. Immediately plate the cells or innoculate a second selective media with the cell suspension. Do not try to pellet the cells through the PEG since this will lessen the transformation frequency, and don’t wait too long to plate or grow up the transformants since the PEG is toxic to the cells. Twenty microliters of cell suspension can give you between 1000-2000 transformants for a healthy yeast strain.
Y153=YJO-Z::Gal1/10 UAS HIS3p HIS3::Gal1/10 UAS + p LacZ
MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112 MEL1
-transform Y153 with “bait” vector (pAS1)+ gene of interest. (pAS1 contains the TRP1 selectable marker)
-check with complete-his + aminotriazole(AT) plates and XGAL overlay test for activation. If no activation proceed.
-inoculate 5 ml complete-trp media with single colony from above.
Grow on 30oC roller overnight or until A600>1.
-use above overnight to inoculate 20mls c-trp grow overday at 30oC..
-use above overday to inoculate to inoculate 200ml c-trp and grow overnight at 30oC. Take A600.
-inoculate 500mls YEPD such that in ~2 generations the A600=0.5 to 0.8 .
(use of YEPD is to increase transformation efficiency however it will result in variable plasmid loss.)
-harvest cells 2.5-5KRPM Sorvall
-wash one time with distilled water ~100mls
-resuspend in 50mls LISORB and incubate at 30oC for 15-30′.
-spin down as above and resuspend in ~625µl LISORB or 325µlLISORB +325µl10%DMSO for later freezing. Hold on ice or at -70oC.
-prepare carrier DNA mix.
Boil 200µl 20mg/ml carrier DNA for 7-10′
add 800µl LISORB (room temp, RT)
mix by pipeting
cool to RT on ice
-Add ~100µl of above DNA mix to 100-200µl cells
-30oC 30′ (optional)
-to 100µl cells + DNA add 900µl 40% PEG3350 in 100 mMLiOAC/TE, incubate 30oC for 30′.
-recovery: pool cells and add to ~100ml complete-his,-trp, -leu shake at 30oC for 1-3 hours; harvest cells and resuspend on ~6ml c-his,-trp,-leu and plate ~300µl per 150mm plate. 20-30 plates used for one transformation, plates=complete media minus his, minus trp, minus leu + 25 mm AT
Efficiency ~5×104 to 105 colonies/µg cDNA.
-for determination of efficiency plate 5µl before and after recovery on 100mm complete-trp,-leu plates.
LISORB=100 mM LiOAC, TE, 1 M Sorbitol.