Talk to Mike.
PCR Method (Microhomology)
Talk to Isis and see []. Plan to screen at least 12 colonies, as this method is known to have a 0.1-5% efficiency.
Plasmid Method (Disintegrators)
We use the disintegrator vectors from EUROSCARF, stored as pCS1439 to 1441.
- 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).
- 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).
- 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.
- 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.