If you want to do a two-hybrid or similar screen start with the Quick and Easy Transformation Procedure to transform your bait plasmid into the yeast strain you will be using. Then use the Standard Transformation procedure to do an experiment to test your library plasmid DNA for transformation yield to determine the scale you wish to use. Finally, use the High Efficiency Library Transformation procedure with the appropriate scale to generate the transformants to screen your library. FAQ
If you have used the Quick and Easy transformation protocol it is likely that your yeast source was too old. Try the Standard protocol with your plasmid as well as the plasmid provided in the kit. This will give you better results. If you have used this high efficiency procedure, it is likely that there is a problem with your SC-minus drop-out medium that selects for the transformed yeast cells. Check the medium recipe against the strain genotype. Better yet use the recipe listed on the Yeast media Page.FAQ
For highest efficiency you should use your cells immediately upon reaching the correct titer and continue until the yeast cells have been plated onto the selective medium. You may want to prepare competent yeast cells that can be stored in the freezer and used at a moments notice. There is a protocol published in Schiestl et al. (1993) which gives reasonable levels of transformed cells. What is the relationship between OD600 and yeast cell density? Each yeast strain is a little different but essentially an OD600 of 0.1 roughly corresponds to 1 x 106 cells/ml.FAQ
You likely have a contamination problem. Carefully bleach your culture to destroy the contaminating bacteria or fungi and then check your medium stock bottle as well as the plate the inoculum was started from. You may find that the liquid medium was contaminated or your plate used to streak out your yeast strain contained contaminants. Check your other media bottles to determine if your problem is with the autoclave step of sterilization. Also inspect your inoculum plate to determine if any colonies have a different color or shape which may indicate a contaminating organism. If your media bottles and inoculum look OK you had better practice your sterile technique or use a laminar flow hood (usually not necessary).FAQ
You probably have an infection in your plate storage cold room. Most cold rooms are cold and dark and ideal for fungus growth. We store our plates in large plastic storage containers in the cold room and every now and then disinfect them with a dilute solutions of bleach (0.3% Na hypochlorite). Inspect your cold storage facility and disinfect if necessary.FAQ
The first thing is to 1) transform your bait or reporter plasmid into the yeast strain and test for auto-activation. If your bait or reporter does not show auto-activation you are ready to proceed. The second experiment that is important before doing a screen is 2) the titration of your library plasmid DNA to determine what size of scale up is required and how many transformants are expected. Grow up your yeast strain as in Protocol 3 in 15 ml of SC-Trp and then sub into 50 ml of YPAD. Do 5 different transformation reactions with increasing amounts of library plasmid DNA (0.0, 0.1, 1.0, 2.0, 5.0 µg). Determine the amount of library DNA that most effectively gives the number of transformants needed to cover your library. (We usually attempt to recover about 20 million transformants) Scale up the transformation to either 30 or 60 X. If necessary the transformation reaction can be scaled up to 120 x.FAQ
YES! For 2 reasons: 1) To more effectively use your library plasmid DNA and 2) to aid in eliminating transformants that take up more than one plasmid. Under conditions of high DNA concentration many yeast cells take up more than one DNA molecule. If the DNA source is a complex cDNA plasmid library then many transformants will contain 2 or even 3 different library plasmids. This complicates the subsequent analysis of positive colonies. I have done yeast transformation in the past and used a 5 min heat shock.FAQ
YES! We have extensively studied the length of the heat shock and found the heat shock time is necessary to obtain a high transformation efficiency. A heat shock of 5 min will not give rise to optimal transformation efficiency using this Kit.FAQ
Some publications have used 10% volume of ethanol or DMSO or a specific concentration of bme or DTT in the transformation reaction. This may help if your transformation reaction is not optimized! We have found that adding these components to the transformation reaction generally reduces the efficiency if you use a 20 to 30 min heat shock . If you reduce the heat shock to between 5 and 10 mins you will end with the same efficiency levels without these components and the longer heat shock. It may pay to run the test experiments to determine the best heat shock for the strain and the conditions you have chosen.FAQ
Unfortunately you cannot. It appears that some bait fusion protein seems to have a detrimental affect on yeast growth. As long as your bait does not autoactivate the reporter gene it may be best to co-transform your bait and library plasmid DNA . The transformation efficiency will be reduced but you should still be able to generate the number of transformants necessary to cover the complexity of your library. Before you begin however, perform an experiment to determine the best ratio of bait and library plasmid DNA and then scale up your transformation to generate the desired numbers of transformants.FAQ
We always use the best water we have available. Cartridge purified water, such as Milli-Q or NanoPure, gives the best results. Double distilled water can also be used, but the rule of thumb is to use the best water you have available.FAQ
Yes, however freezing dramatically reduces the transformation efficiency. Schiestl et al. (1993) gives a protocol for the production of the frozen competent yeast cells, however the transformation efficiency is only in the range of 1 x 104 transformants/µg.FAQ
Plasmid DNA does not need to be extensively purified for transformation
into yeast. Plasmid DNA containing E. coli RNA transforms just
as effectively or in some cases even better than the same preparation
after removal of the RNA (Gietz Lab, unpublished observations).
FAQ
One person wrote in telling of an individual in the laboratory
that followed the protocol but still got no Transformation! After
observation it was determined that the problem was the plating
procedure. This individual was over spreading the transformed
yeast cells onto the plate (2-3 minutes of continual spreading
until the fluid was fully absorbed by the medium). This action
killed the transformed yeast cells resulting in no transformation.
When spreading be careful to allow your sterile glass wand to
cool sufficiently prior to spreading yeast inoculum. Then spread
the inoculum evenly over the plate with a minimum number of strokes.
Allow the medium to absorb the fluid (will take 2 to 5 min depending
on how dry the plates are) and then incubate until colonies appear.
This should be 2-4 days depending on the medium recipe used.
FAQ
This has been the most popular question of late! The answer is to use a good vortex and just keep it mixing at max until it is resuspended. When we are doing standard transformations in a 1.5 ml microfuge tube we use two tubes opposed to each other so they knock into each other. This helps dislodge the pellet from the wall of the tube. For large scale transformations try and get vigorous mixing with the tube at an angle so that it also vibrates as well as mixing. Wolfgang Lankes from the Max Delbrueck Center for Molecular Medicine, wrote me with a good solution to the resuspension problem. He uses a rough surface such as a wire test tube basket to quickly run the end of the tube over a couple of times. He says this works very well to resuspend the pellets into TRAFO mix or even sterile water at the end of the TRAFO reaction. Be careful not to use a rough surface as it could damage the tube which may cause it to fail during centrifugation FAQ
This is probably one of the most asked questions! Reasons for transformation variation!
We dissolve our carrier DNA in sterile TE and handle all operations with sterile technique. Then we consider our carrier DNA sterile after we place in a boiling water bath for 5 mins. We have had NO sterility problems that could be traced back to the carrier DNA!!!!
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