Smolke:Protocols/Chromosomal Modification

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Knockouts Talk to Mike. Insertions PCR Method Talk to Mike. Plasmid Method Overview We use the disintegrator vectors[1] from EUROSCARF, stored as pCS1439 to 1441. Protocol Clone your gene of interest into the appropriate integrating plasmid Prep the resulting plasmid and digest with an enzyme that only cuts the YIp-In. E.g. NruI for 1441, AleI for 1440. Transform your desired yeast strain using standard chemical methods. Select for growth on -URA. Patch colonies from the -URA plate onto a new -URA plate (both to save and to verify that they really are URA positive). Verify integration, either by PCR or by functional tests. Realize that you might have integration, but at the wrong locus. Select a strain with the correct integration. Grow in YPD for ~2 days, back diluting in the morning and evening. Plate cells on 5-FOA plates, being careful not to saturate the plate. If you have too many cells on the 5-FOA plate, restreak to single colonies on a new 5-FOA plate. Patch colonies from the 5-FOA plate onto a new 5-FOA plate (both to save, and to verify that they really are URA negative). Verify that the desired cassette is still integrated, either by PCR or by function. To further confirm, you can check that the cells cannot grow on the appropriate plates (-Lys/-Met/+5-FC). Notes Anecdotally, I had about half of my first round of colonies (Step 3, above) integrate into the correct locus. A strain with integration into the wrong locus cannot recombine out the URA marker. I was integrating a fluorescent protein, so identification of the correct locus was straightforward - cells with the correct integration showed similar fluorescence histograms. Cells with the wrong locus showed a different mean fluorescence. If you're integrating a fluorescent protein, checking for integration is easy - just scrape cells off a plate, resuspend in PBS, and run on the flow cytometer. After removing the marker (step 9 above), <10% of the resulting cells still contained the integration cassette. The rest had just recombined out the original plasmid (recombination through YIp-In rather than the desired YIp-Out). As mentioned, checking for fluorescent cassettes is easy. Otherwise, screen by colony PCR or check for loss of the wt integration locus (so look for lys- colonies if using pCS1441). Again, anecdotally, integration into the lys2 locus (pCS1441) gave similar mean fluorescence to a cen4 centromeric plasmid (pCS2), but a much reduced variation in expression (CV decreased by ~2x). References Sadowski I, Su TC, and Parent J. Disintegrator vectors for single-copy yeast chromosomal integration. Yeast. 2007 May;24(5):447-55. DOI:10.1002/yea.1469 | Güldener U, Heck S, Fielder T, Beinhauer J, and Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996 Jul 1;24(13):2519-24. DOI:10.1093/nar/24.13.2519 | Gueldener U, Heinisch J, Koehler GJ, Voss D, and Hegemann JH. A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res. 2002 Mar 15;30(6):e23. DOI:10.1093/nar/30.6.e23 |