yeast transformation efficiency issues - (Sep/20/2006 )

Hello:
I'm fairly new to this forum.
I am using AH109 strain for Y2H screening and my transformation efficiency is very poor, even following Clontech's protocol (LiAc/PEG) with DMSO. Has anyone have any similar problems with this particular strain or can anybody recommend of a better strain to use for Y2H? Thanks.

---

Hi,
In our lab we follow a modified version of the clontech protocol (although our bait is in AH109 and the library is in Y187). One thing we found to help improve our transformation efficiency was to make sure that the PEG/LiAc was mixed extremely well before adding to the transformation mixture. Are you transforming your library DNA into your bait strain? If you could tell me a little more about your procedure, maybe I something will jump out......

---

---

Hi shurgood,
Thanks for the reply.
I am transforming the library into AH109 that already contains the bait plasmid (sequential transformation). I tried to optimize it by testing different heat shock times and there was no improvement. I initially grow the bait strain in SD/-Trp until the titer reaches 10^7 then subcultured in the same medium to 10^6 cells/ml until the titer reaches 10^7 cells/ml. I have tried subculturing them in YPAD, and/or adding an hour in YPAD after heat shock but did not see any improvement.
I'm curious what efficiency did you get.
yeast

---

I usually grow the bait strain (AH109) in 2X YPAD. I'm not in the lab right now, but I will post some of my transfomation efficiencies tomorrow when I get a chance. Is there a chance your bait could be toxic to the yeast? Here is a modified protocol that we follow in our lab:

High Efficiency Transformation

(adapted from Clontech User manual PT3529-1)

1. Innoculate one colony of yeast in 50 ml appropriate dropout media. Incubate overnight, shaking (240rpm) at 30 degrees C. Take OD 600 in morning and dilute to OD 600 of 0.23 in prewarmed selective media (or 2xYPAD if no selection needed). For Y2H transformations into bait strain, grow up 150 mls total.

3. Incubate shaking at 30 deg/C for 3-5 hours until OD 600 is between 0.6-0.8

4. Pellet cells (700 x g, or 2000rpm, for 5 min). Remove supernatant and wash cell pellet in sterile water. Pellet again and remove supernatant.

5. Resuspend pellet in 2.5 ml TE/LiAc (550µl 10xTE; 550µl 1M LiAc; 3.9ml dH2O) Divide into two 1.5 ml microcentrifuge tubes and pellet at 10,000rpm 1 min.

6. Remove supernatant and resuspend each pellet in TE/LiAc solution. For transformations into bait strain, resuspend each pellet in 1 ml (including pellet, so add slightly less).

High efficiency transformation

Place tube of ssDNA on heat block, then boil 3 min and place on ice until cold. Make LiAc/PEG solution (make fresh! : 8 ml PEG; 1 ml 10xTE; 1 ml 1M LiAc)

per sample
library DNA 2-3 µg
ssDNA 5 µl
yeast cells 60 µl
LiAc / PEG mix 250 µl


11X mix
library DNA varies
ssDNA 55ul
yeast cells 660ul
LiAc/PEG mix 2.75ml


Mix in a 15 ml conical tube. Use 3 tubes of 11X mix each for a two-hybrid screen. For each conical tube, add each item in the order indicated, then vortex immediately. It is important to vortex well so that the solution is homogenous.

Incubate 30 deg/C for 45 minutes. Mix cells every 15 min. Add 176 µl DMSO to each conical tube. Mix well by inverting up and down several times. Place tube in 42 deg/C bath for 20 min. Mix cells every 10 min.

Pellet cells (700xg 5 min) and remove supernatant. Resuspend in 0.9% NaCl solution and plate. For 33x screen, resuspend in ~4.8 ml total volume and plate about 150 µl per plate, onto 30 plates.

added:
our efficiencies range between 10^6-10^9 (which is good enough for coverage for the libraries we use, we try to get at least 3 fold coverage of the library....)

---