Hello to all you Buddies striving to get the most out of yourtransformations!!!! There have been a number of articles posted to this board discussingtransformation efficiency. How do I get more etc. What is the best method?Let me start by saying that all strains are not created equal when it comesto transformation efficiency when using the different methods! I havefound that a strain that transforms with a medium efficiency withLiAc/ssDNA/PEG transforms better with electroporation. Again If I take allthe strains I normally use in my lab and compare there is a wide differencein the TRAFO efficiency we see! There are a few things one can do tosqueese the most TRAFOs out of a strain with the LiAc/ssDNA/PEG method. 1. Make good single stranded salmon sperm Carrier DNA! The key is to keepthe DNA as large as possible (MW) but still be able to handle it afterboiling and quick cooling! As most know that if you dissolve largemolecular weight DNA in TE at 10 mg/ml and boil and quick cool you get avery stiff gel. Not good for adding to a TRAFO reaction. Therefore I make100mls of 10 mg/ml and once insolution I sonicate for 30 sec with a largehorn and then test 1 ml of it for the 5 min boil quick cool test. Usuallyit takes 2 -3 30 sec blasts to get the DNA small enough that it will stay aliquid after the boiling and quick cooling. I have recently found thatsome batches of salmon spern DNA from Sigma donot need to be phenolextracted to give very good transformation. If you have having problemswith the 10mg/ml solution make one at 1 mg/ml. This solution is easier tohandle but increases the volume of the cDNA added to the TRAFO reaction. Ifind that good carrier DNA is very important for good TRAFO efficiencies. Fatima mentioned that dirty DNA (DNA with RNA from the plasmid prep) isbetter the clean stuff. Well She is right. In our first paper RobertSchiestl and I showed that RNA can also be used as a very effectivecarrier. So dont go to great lengths to clean up your DNA! (Which themammalian transfections require) RNA contamination actually helps.2. To get the best TRAFO efficiencies out of your strain one shouldoptimize a number of variables. The heat shock is very important. Mostpeople dont like to give a 15 or 20 min heat shock at 42¡ but believe memost strains need it. If your strain is heat sensitive, reduce thetemperature to 40¡ or 37¡ but do the experiment to see what duration givesyou the best transformation. You can add DMSO or Ethanol as somereferences have eluded to but we have found that if your heat shock isoptimal you get no enhancement by adding these things in the strains wehave tested. Try it though, it may work for you!3. Optimize the amount of carrier DNA for your strain. Some carrier prepsdiffer and we have have seen some strains that need 75 ug instead of 50 forthe best TRAFO.4. Growth conditions are important. Make sure that your culture isactively dividing! Most strains transform best with LiAc after they havegone through 2 division. We subculture to 5 x 10^6 cells/ml and let growto 2 x 10^7 cells per ml. This takes 3 - 4 hrs and is well worth the waitif you are trying to eek out all the TRAFOs you can. 5. The % PEG in the TRAFO reaction is fairly important as the peak is quitenarrow. If it gets too high TRAFOs go down dramatically! We had a twomonth period in my lab were we lost the ability to get good TRAFOs. Wetracked it down to PEG which was stored with a loose lid and was at aconcentration higher than 50% thus reducing the TRAFO efficiency. Followthe TRAFO recipe exactly and make sure your PEG is 50% weight by volume!!! Thats 50 gms of PEG 3350 MW in a final volume of 100mls.6. If you need to put a library into a strain along with anotherplasmid like in the two hybrid system of Fields and Song then it is best totransform in the bait plasmid first (pMA424 or pAS or equivalent). Thengrow a 10 ml culture of this strain selectively, to keep the bait plasmidin the cells, in SC-HIS or TRP to give about 1-2 x10^7 cells/ ml. I thendump this 10 mls into 40mls of warm YPAD and grow for 2 generations. Thereis usually negligable plasmid loss in two generations. Transform as usualand plate onto double omission medium. Contrary to some opinion we havefound that transformation of two plasmids from a mixture is only 30 -40%that of a single plasmid.7. Some strains never give good TRAFO by LiAc/ssDNA/PEG. With these Iwould go to another method as suggested by others. The Electroporationmethod of Manivasakam and Schiestl,1993 NAR 21:4414-4415 is a good one. 8. Another thing I have found that make a difference is the pH of theselective medium you are plating on. We routinely adjust to pH 6.5. If itis too acidic this appears to decrease transformation efficiency. Anotherimportant point that some forget is to keep your SC-minus medium away fromlight. We have traced poor TRAFOs and just plating efficiency to platesthat have been expose to 24 hrs of fluorescent light. Cover those platewith a box when drying and keep the cold room light off (if thats where youstore them) or else store them in covered container. YPAD plates dont seemto be affected but SC-minus medium is seriously affected.REFERENCES for all this stuff areSchiestl and Gietz 1989 Curr Genet 16:339-346Gietz and Schiestl 1991 Yeast 7: 253-263Gietz et al 1992 NAR 20 1425Schiestl et al 1993 Methods:A companion to methods in Enzymology 5:79-85There are also another other book chapter coming out discussing how to TRAFO to the MAX Watch for: Molecular Genetics of Yeast (Oxford University Press) Due soonAlso another is planned from CSHLs.Anyone that has specific questions about TRAFO please feel free to drop mea message to the address below!GOOD TRAFOs!!!!!!!! Dan Gietz**************************************************************************R. Daniel Gietz Ph.D.Department of Human GeneticsUniversity of Manitoba770 Bannatyne Ave, Rm250Winnipeg, Manitoba, Canada R3E0W3Tel(204)789-3458 Fax 204-786-8712 Email:GIETZ at BLDGHSC.LAN1.UMANITOBA.CA*****************************************************************************
联系客服
