Yale Genome Analysis Center, Yale University http://ygac.med.yale.edu/mtn/reagent/avail_reagents/lacZ_LEU2_info_p.stm
| TR |
Tn3 terminal inverted repeats |
| lacZ |
5'-truncated lacZ gene encoding beta-galactosidase |
| LEU2 |
LEU2 gene from S. cerevisiae |
| amp |
Encodes beta-lactamase |
| loxP |
lox site, target for Cre recombinase |
Uses: Gene disruption, analysis of gene expression, immunodetection of beta-gal fusion protein.
In more detail: mTn-lacZ/leu2 was constructed by Siefert et al. (1986). It can easily be inserted at mutiple sites in a given gene. The mutagenized DNA is then transformed into yeast, where it replaces the chromosomal locus by homologous recombination. The transposon insertions create a pool of insertion/disruption alleles. Insertions that generate in-frame fusion of the coding region to lacZ can be used to monitor and quantify gene expression, via assays for beta-gal activity. The fusion protein can also be immunodetected using antibodies directed against beta-gal.
The accession for mTn-lacZ/LEU2 is U35112
A kit for mutagenesis of a yeast gene with mTn-lacZ/LEU2 is available.
Protocols for shuttle mutagenesis/epitope-tagging of a yeast gene with mTn-lacZ/LEU2
Please read this whole document before you start!
Shuttle mutagenesis
- Clone fragment into vector pHSS6 (from strain R1123; map given below).
- Delete as much of the polylinker as possible as sometimes transposon 'hot-spots' into it. Select transformants on LB Kan40.
- Transform this plasmid into competent cells of R1236/B211.
- Transfer F::mTn-lacZ/LEU2 into cells by mating with strain B198
- Grow strains overnight with antibiotic selection (Amp50 for B198).
- Subculture 1:100 in fresh medium (no antibiotics). Grow at 37oC to early log phase (when cell swirls are visible). The recipient strain (B211) can be denser than the donor.
- Mix 200 ul of each strain. Incubate at 37oC without agitation for 20 min to 1 hr. Plate as 100 ul aliquots onto LB Amp50 Kan40 Cm34.
- Grow 1-2 days at 30oC. Now have cointegrates. (Set up strain R1230 in Sm50 overnight).
- Mate to strain R1230 to resolve cointegrates
- Elute colonies from plates: put 2 mls of LB on the plate, scrape off the colonies with a speader. This is your eluate. You should have several thousand colonies at least.
- Dilute overnight culture of strain R1230 1:100 without antibiotic. Dilute eluate to roughly same density.
- Grow and mate as before.
- After mating for 20 min to 1 hr, plate 100 ul aliquots on LB Amp50 Kan40 Sm50 and grow overnight at 37oC.
- Do the Control: Spot the starting strains onto this media.
- Rescue resolved DNA from this strain
- Elute your colonies off in LB. Again, you should have thousands. Dilute some eluate in LB Amp50 Kan40 to give an almost saturated density. Grow at 37oC for a few hours.
- Isolate DNA by miniprep. (We do a standard 1-2-3 alkaline lysis but use 150 ul of 7.5M NH4Ac as solution III, and 270 ul of isopropanol to precipitate. This removes most of protein (avoiding phenol) and RNA, giving a very small clean pellet. Still, there are nucleases so we keep everything on ice).
- Transform about 1/10 of minprep into a regular recA endA cloning strain (eg DH5). Plate on LB Amp50 Kan40.
- Transform into yeast selecting for LEU2
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