The TRAFO FAQ


Frequently asked Questions

  1. What transformation protocol do I use?
  2. I followed the protocol and got no (zero, nada, none, zip) transformants. What's wrong?
  3. Can I store the Competent cells for any length of time before using them?
  4. My yeast culture really stinks! What is the problem?
  5. I did a transformation and got a lot of fungus all over my plates. What can I do?
  6. What are the steps I need to perform before doing a screening?
  7. Is it really important to do the transformation experiment with increasing amounts of library DNA?
  8. Do I really have to heat shock for 20 to 30 min.?
  9. Some people add b mercaptoethanol, 95% ethanol, DTT, or DMSO to their transformation reactions. Will this increase the efficiency of your method?
  10. We have transformed our bait plasmid into yeast and it grows poorly. Can we do anything to make it grow better?
  11. Is the quality of water critical for transformation?
  12. Can the yeast cells as prepared in protocol 2 or 3 be frozen? If so, how and how well do they transform?
  13. Does it matter if your plasmid preparation contains RNA?
  14. Plating method affects transformation yield. What NOT to DO!
  15. How do I resuspend my pellet of cells into the TRAFO mix that contains PEG? I really have a hard time with this step!
  16. I cannot obtain the transformation levels that you list on this site! WHY?
  17. What variables do i test to get optimal transformation efficiency?
  18. How do I sterilize my carrier DNA?


  1. What transformation protocol do I use?

      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

       

  2. I followed the protocol and got no (zero, nada, none, zip) transformants. What's wrong?

      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

       

  3. Can I store the Competent cells for any length of time before using them?

      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

       

  4. My yeast culture really stinks! What is the problem?

      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

       

  5. I did a transformation and got a lot of fungus all over my plates. What can I do?

      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

       

  6. What are the steps I need to perform before doing a screening?

      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

       

  7. Is it really important to do the transformation experiment with increasing amounts of library DNA?

      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

       

  8. Do I really have to heat shock for 20 to 30 min.?

      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

       

  9. Some people add b mercaptoethanol, 95% ethanol, DTT, or DMSO to their transformation reactions. Will this increase the efficiency of your method?

      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

       

  10. We have transformed our bait plasmid into yeast and it grows poorly. Can we do anything to make it grow better?

      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

       

  11. Is the quality of water critical for transformation?

      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

       

  12. Can the yeast cells as prepared in protocol 2 or 3 be frozen? If so, how and how well do they transform?

      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

       

  13. Does it matter if your plasmid preparation contains RNA?

      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

       

  14. Plating method affects Transformation yield! What NOT to DO!

      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

       

  15. How do I resuspend my pellet of cells into the TRAFO mix that contains PEG? I really have a hard time with this step!

      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

       

  16. I cannot obtain the transformation levels listed on this site! Why?

      This is probably one of the most asked questions! Reasons for transformation variation!

    1. Transformation efficiency has genetic variables. There is dramatic variation in the transformation efficiency of amoung strains currently being used in the general yeast community. Not all strains will give 3 million transformants per microgram of plasmid DNA even if you optimize all the variables. It is best to start with a good transforming strain and then optimize.
    2. Cell Growth is very important. Cells must go thru at least 2 divisions before they become fully competent for transformation. They remain that way for another 1 division. Be sure that you are using a rich media like YPAD and grow the cells from 5.0 x106 cells/ml to at least 2.0 x107 cells/ml.
    3. Media is very important. Ensure you are using a "Drop out" media when plateing your cells. See the Media Page, accessable from the front Trafo page. Ensure that you titrate the pH of you media to 5.6 before autoclaving. We have done the experiment and found that this pH promotes the highest efficiency. YPAD should be titrated to a pH of 6.0. In addition DO NOT expose your "Drop out" plates fluorescent lighting for any length of time. Some bulbs (not all) cause reduced plating efficiency ! (dont know why but it does). We store all our plates under cardboard boxes at room temperature and in a dark cold room.
    4. Ensure that all your reagents are top quality and made correctly. The carrier DNA is an important reagent. Make it according to the instruction on this web site! Do not use the older methods. In addition the PEG reagent must be at the correct concentration. This is very important. If you are having a hard time with TRAFO efficiency, vary the amount of PEG added to the Trafo reaction. You may find that your PEG concentration is a bit off and needs more or less volume to reach optimal transformation efficiency. OK, I am going to be a shameless self promoting capitalist here and tell you if you need help with the reagent part of transformation consider buying the GIETZ Lab Yeast Yransformation Kit. I make and test them myself and know they work! (See the front page for details!) Sorry about that! It is just a royal pain see all these companies (you know the ones) making money off of the technology I developed! FAQ

       

  17. What variables do I test to get optimal Transformation efficiency?
    1. The easiest variable to test is the heat shock length. Test various times from 15 min to 60 min heat shock at 42oC. Ensure you plate duplicate plates to ensure good reproducibility.
    2. The next variable to test is amount of carrier DNA in the TRAFO reaction. Some yeast strains need a bit more carrier DNA. Do a set of experiments to determine the best DNA amount to add to the transformation reaction.
    3. The next variable to test is amount of PEG in the TRAFO reaction. Some PEG stock solutions are not exactly correct due to the viscosity of the solution. Do an experiment to determine the amount needed to get to optimal transformation efficiency. Remember the optimal concentration is 33% w/v.
    4. If you still can't get good transformation, get a hold of a yeast strain/plasmid combination that gives good transformation so you have a bench mark! FAQ.

       

  18. How do I sterilize my carrier DNA?

      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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