Direct transfer of plasmid DNA from yeast to E. coli by electroporation Summary. This procedure describes transfer of plasmid DNA from yeast strain EGY48 to E. coli KC8. It was adapted from one provided by Karen Clemens in Peter Howley's lab at the NIH. The idea was originally published by R. Marcil and D.R. Higgins, NAR 20, 917 (1992). 1. Prepare electrocompetent KC8 cells. Centrifuge cells, wash, and resuspend the final cell pellet in water resulting in an OD600 of 100. A rule of thumb here is to resuspend cells processed from a 1 liter culture (OD600 = 0.55) in 1 ml water. Dilute 5ul of the cell suspension to 1ml with water and measure the OD600. Add more water to the cell suspension (if necessary) to get to OD600 = 100. 2. Distribute the electrocompetent KC8 cells in 65 ul aliquots into ice cold eppendorf tubes. Scrape off yeast from a streak colony (about 10 ml of cells) grown on an Xgal Gal/Raff plate (other plates probably work, but we haven't tried them yet). Resuspend the yeast cells by swirrling the stick used for scraping off the cells in the KC8 suspension, try to get an even distribution of the two kinds of cells (but don't vortex, which might break the fragile water-suspended E. coli). 3. Transfer the mixture to an ice cold 0.2 cm electroporation cuvette (Biorad) and use the electroporator to deliver two pulses. The first one is to release the plasmid from the yeast cells whereas the second gets it into the E.coli cells: 4. Shock the cells. 1st pulse setting: 1500 Volts, 25 mF, 100W. Expected time constant: 2.2 - 2.4 msec. After this pulse, take the cuvette out, and place it on ice for at least 30 seconds. Meanwhile change the settings for the second pulse. 5. Shock the cells again. 2nd pulse setting: 2500 Volts, 25 mF, 200W. Expected time constant: 4.2 - 4.8 msec. 6. Take the cuvette out, add 1 ml LB medium and transfer the suspension back to the original eppendorf tube. Incubate for 45 min at 37oC with light agitation. 7. Spread 150ul of the suspension onto E. coli plates. These should be 1XA or M9 minimal medium. To rescue standard interaction library plasmids, which carry a TRP1 selectable marker that complements the trp- phenotype of the E. coli strain, the plates should lack tryptophan but contain ampicillin (100 ug/ml), leucine, histdine, and uracil at standard concentrations (Current Protocols in Molecular Biology, Greene Publishing and John Wiley and Sons, New York, 1987-1994). Keep the rest of the transformation mixture at 4oC in case you need it for a repeat. During an interactor hunt EGY48 contains three different plasmids, all of which confer amp resistance if transferred into E. coli. In order to select for the interaction library plasmid ("prey plasmid") Minimal Medium lacking tryptophan is used. To rescue plasmids that carry other selectable markers, use minimal plates that contain amp but lack the appropriate nutritional supplement 6. Incubate the plates at 37 oC until single colonies are easy to pick (about 30 h). You should get between 50 and 200 colonies. Don't worry about a slight yeast background on the plates. Pick a single KC8 colony and inoculate it into 1.5 - 5 ml LB Amp (100 mg/ml) or Minimal (trp-, leu+, his+ ura+ Amp), grow at 37 oC, and harvest at an appropiate OD to prepare miniprep DNA. Save the plate in the refrigerator. Strains sometimes contain multiple library plasmids. Should you think this may be occuring, either because your transformation was done at higher DNA concentrations than recommended, or because subsequent introduction of the library plasmid into the original selection strain does not reproduce the interaction phenotype, perform minipreps from ~6 transformants, analyse cDNAs in the libary plasmids by restriction digestion or by PCR, and save representatives of each cDNA class for further analysis. 6 December 1993 modified 31 January 1994 Timm Jessen, and Roger Brent 617 726 5956 617 726 6893 (fax) jessen@opal.mgh.harvard.edu brent@opal.mgh.harvard.edu