YKO pool transformation using CEN of 2µM pRS vectors
Last updated: November 21, 2003 by Siew-Loon Ooi

 

1-1.5 µg pRS416 into 30 OD of cells would yield about 3x 105 colonies.

 

Total OD cells Cells Volume Total OD/µl HSDNA Transforming DNA PEG DMSO
30 150µl 0.2 OD/µl 10µl 1-1.5 µg 444µl 67µl

 

Outgrowth of YKO mutant pool


1) Thaw 1 tube of YKO mutant pool. Each vial contains 1 or 2 ml of ~14 OD600/ml of pooled YKO mutants, and is sufficient for 5 routine transformations.
2) Dilute cells in YPD in a 1 liter flask to the desired starting OD600 of ~ 0.125-0.15 OD/ml. For 2 ml of 14 OD600/ml YKO mutant pool, this would require ~ 280 ml of YPD.
3) Culture cells at 30¾C with shaking at ~ 250 rpm until the OD600 reaches 0.5 (2 generations) OD/ml. This usually takes about 5-6 hours.
4) Monitor OD600 using spectrophotometer.
5) Write down the volume and the OD600 of cells. This information will be used to adjust the competent cells to an OD600 of 0.2 OD/µl later.

Preparation of competent cells

6) Aliquot cells into six or seven 50 ml conical tubes.
7) Spin cells down at 1500-2000 RPM for 5'.
8) Wash once in water.
9) Wash once in 0.1M LiOAc.
10) At this step, adjust the OD600 of the competent cells to 0.2 OD600/µl by resuspending the cells in (Total OD600 x volume/ 0.2 OD600) µl of 0.1M LiOAc. This usually requires 750-1000 µl of 0.1M LiOAc.

 

Transformation


11) Boil Herring Sperm DNA (10mg/ml, HSDNA) at 100¾C for 10'. Set on ice.
12) Add 20 µl HS DNA as carrier to sterile eppendorf tubes in which transformation will be carried out.
13) Add the appropriate amount of transforming DNA.
14) Add 150 µl of 0.2 OD600/µl competent cells to each tube.
15) Add 444 µl PEG (45.4 % PEG3350/.1 M LiOAc) to each tube. PEG3350 and LiOAc stock solutions are usually mixed together just before transformation. For each reaction, add 400 µl of 50% PEG3350 and 44 µl of 1M LiOAc to prepare the PEG3350 solution.
16) Incubate at 30¾C on a rotor for 30'.
17) At this point, the reaction should contain:

Reagents Volume (µl) Final [ ]
HSDNA (10 mg/ml) 10 0.165 µg/µl
DNA (1-10 µg) 1-10  
Competent cells 150 0.05 OD/µl
45.4% PEG3350/0.1M LiOAc 444 33% PEG3350/.1M LiOAc
Total Volume 604

 


18) Add 67 µl of DMSO to each tube. The final concentration of DMSO should be 10%.
19) Heat shock at 42¾C for 15'.
20) Spin at top speed for 10".
21) Wash once in water.
22) Resuspend in 4.3 ml water.
23) Plate 400 µl on each of 10 large (150 mm) plate.
24) Also plate cells at different dilutions to monitor transformation efficiency.

Comments


Amount of transforming DNA volume: 30 OD600s of competent cells from MATa haploid YKO pool transformed with 1-1.5 µg of pRS416 should yield about 3 x 105 colonies. To obtain 106 colonies, more plasmid DNA (5 to 10 µg) should be used.


Volume of transforming DNA: If the concentration of the input DNA is too dilute, try to concentrate it to 1-5 µl for each transformation by ethanol precipitation.


Number of competent cells used for transformation: Initially, 30 ODs of cells were used per transformation reaction. But now, a total of 15 ODs of cells is used per transformation, and the transformant yield is comparable.


Amount of HSDNA: Initially, 10 µl of 10 mg/ml of HSDNA is used, but this was increased to 20 µl of 10 mg/ml of HSDNA. Preliminary results suggest that this increases the transformation efficiency slightly when more transforming DNA is used.