Yeast Competent Cells - (Feb/06/2008 )
That would be great, the more help the better.
Below is the protocol for the preparation electrocompetent yeast cells
1.Grow cells to 1X10^8 or OD600 of 1.2-1.3.
2.Spin down cells at 4,000 rpm for 5 min
3.Wash pellet in an equal volume of ice-cold water.
4.Wash in 1/2 volume of ice-cold water
5.Wash in 1/25 volume of ice-cold 1M sorbitol
8.Resuspend cells in 1/200 original volume of cold 1M sorbitol (at this stage you can prepare 50 μl-aliquots of the cell suspension, freeze them in dry-ice and store them at -80º C).
10.Mix 50 µl of cell suspension with no more than 5µl DNA (in low ionic strength buffer).
11.Immediately tap gently cell/DNA suspension to the bottom of a 0.2 cm cuvette and pulse at 1.5 kV, 200 Ω, 25 µF (pulse time: 5 millisec).
12.Immediately add 1 ml YEPD/ 1M sorbitol and allow cells to recover at permissive temperature for one hour.
13.Spin down cells and resuspend in 1ml 1M sorbitol.
14.Plate on selective medium (use a glass spreader to spread the cell suspension but it is really important not to disturb the cells much)
Thank you for that , i'm trying a couple more things then that may well be my next attempt.
I have the results from my last transformation and cleaning the plasmid did the trick (frequencies up to 10^5). I just did 2v ethanol and 0.1v 3M NaAc at -80C for an hour, spun it and followed with a 70% ethanol wash.
Other things: I make YPDA by autoclaving the yeast extract and peptone together in a bottle and autoclaving the glucose with some water in a separate flask. I have a separate autoclaved stock of 0.2% adenine hemisulfate which I add to the YPD after the YPD has been autoclaved. Some recipes may say to add the adenine hemisulfate (solid) to the YPD before autoclaving, but there are precipitation issues... so make a separate stock of adenine hemisulfate and either autoclave or filter sterilize.
One of the biggest challenges I faced when learning how to do the yeast transformations was being able to plan the experiments so that the cell cultures would be ready at decent times (i.e. NOT 3am). I've tried a lot of parameters and found what works for me- I follow the instructions that come with the Clontech kit. To begin, I inoculate the first 3ml cultures with a single yeast colony. I grow that 8 hours and measure the OD to make sure that the culture hasn't entered stationary phase. Then I grow 4 separate 50ml cultures which contain either 5, 10, 20 or 40ul of inoculumn- this is the only change I've made to the Clontech protocol. I use whichever culture has reached an OD600 of 0.15 earliest, usually "20ul" will be ready within about 20-22 hours, I then spin down that flask and transfer that to 100ml YPDA and that will grow to an OD600 of 0.4 within 4 hours (like the manual says).
I shake the cultures pretty hard (about 300 rpm). I also grow cultures in flasks much larger than the manual says - I use 500ml for 50ml cultures and 1000ml for 100ml cultures.
Hope this helps...
i have just made fresh media where i add the dextrose after autoclaving and am giving the transformation a go today. i quantitated the plasmids that came with the kit on a nanodrop and all 260/280 ratios were above 1.85 but some of the amounts were less or more than stated so I am going to adjust accordingly.
I know what you mean about the culture timings my first couple of goes I was at work so late! After growing my first culture I seed my 50ml at about 6000cells/ml and find the next morning im at about 0.2 so can get straight on (I base my calculations on 0.1 abs at 600nm being equal to 1x10^6cells/ml, think i got that from the TRAFCO siet),seems to work.
How is best to spread cells, I have been using a glass spreader but am going to try beads as well.
Anyway thanks for the tips,stay in touch would be good to know how you are getting on.
just had a peek at my plates and looks like im going to be in the 10^5 range for my transformation efficiency although i only have approx 20 colonies on the 1/100 plate protocol reckons there should be at least 30. Im going to plod on with the other control plasmids and see how it goes.
thanks for your tips, looks like they helped.
Steve- Do you mean that you're using 300ul of a OD600 0.1 culture to inoculate the 50mls of YPDA?
I haven't done anything special with spreading, I just use a glass spreader...
20 colonies of the 1/100 dilution sounds good. Did you clean the plasmid? Go try your controls!
General Forum question-
One thing I have recently had problems with is satellite colonies forming on my plates after 3 days. I can definitely tell my transformants from the background, but it is still not ideal. Does anyone know the cause or the remedy?
For my last experiment I grew a single colony for eight hours and measured the OD600, it was 0.697. I took this to equal 6.97x10^6 cells/ml. I then added 43ul of this culture to the 50ml YPDA (this gives 6000cells/ml) and after 14 hours the culture had an OD 0.273 so I could start as soon as I came in to the lab.
This is based on OD 0.1 = 1X10^6 cells/ml and a doubling time of approx 1.5 hours, I have done no studies on these numbers I just got them from reading but they seem to work and save me some time.
I have not seen any satellites on my plates and cant think of anything at the moment but i'll keep thinking !
I've asked some people with experience with yeast two hybrids- satellite colonies seems pretty common. I can definitely tell the difference between satellites and transformants and I don't get satellites on -His plates.
I'm planning to transform the library into cultures which already contain the bait plasmid. I'm testing the transformation frequencies for that right now.
I just finished making cDNA with the Matchmaker kit and it was really really easy! Finally something went smoothly on the first try...
Glad things seem to be going well for you. Im about to start testing some of my bait constructs for background expression then move on to some preliminary screens. You are right about the cDNA it ws easy, certainly more straight forward than the rest of this project !
I'm going to co-transform my baits with the cDNA and the linearized pGAD vector, did you say you have already transformed your baits and are then doing your library ? Wondered if there was a reason for this?
Anyway best of luck