Methods for use with the mTn-lacZ/LEU2-mutagenized library

The following protocols are included:

• DNA">Making library DNA from the DNA we send you
• Transforming yeast with DNA from the insertion library
• Screening for gene expression using lacZ fusions
• Identification of the genomic site of transposon insertion
• Transferring the disruption allele to other strains

DNA>Making library DNA from the DNA we send you

The library is distributed as individual pools in the form of DNA. You will be sent about a microgram of each pool available. Transform a suitable amount into E. coli (any strain suitable for making plasmid preps). Select transformants with 40 ug/ml kanamycin and/or 50ug/ml ampicillin. Obtain 50,000 colonies for each pool. Elute colonies from plates in LB; make a -70C stock of this eluate. Dilute eluate into LB plus antibiotic to give a culture with an almost saturated density. Grow at 37C for a few hours. Make miniprep or midiprep DNA.

Transforming yeast with DNA from the insertion library

OVERVIEW: Mutagenized DNA from the library is excised from the bacterial vector. It is then transformed into a leu2 strain of yeast. This procedure is outlined in this figure. Use of a circle-zero strain will prevent recovery of insertions in the 2-micron plasmid. The best strategy is to screen a few thousand transformants from each pool. Screening 30, 000 transformants should give you 95% coverage of the yeast genome.

To minimize double integrants, transformations should contain the lowest amount of DNA practicable. We therefore recommend that a pilot experiment be performed to determine transformation efficiency of the strain, and conditions then be scaled up as appropriate. The pilot protocol given below uses a modified version of the method of Chen et al. (1992). You should use whatever transformation protocol works best in your hands.

1. Plasmid DNA from pools of the mTn3-mutagenized genomic library is digested with NotI. A 2.1-kb band from the vector should be apparent, together with a range of bands in the 8-kb region.
2. A 10-ml culture of the yeast host strain is grown to a density of 10⁷ cells/ml (O.D. 600 of 1). Use of such logarithmically-dividing cultures increases transformation efficiency.
3. Cells are pelleted and washed once with 5 volumes of One Step buffer (0.2M LiAc, 40% PEG 4000, 100 mM beta-mercaptoethanol). This wash is especially important when culture volumes are increased.
4. Cells are resuspended in 1 ml of One Step buffer containing 1 mg of denatured salmon sperm DNA. 100 ul aliquots of this suspension are then added to tubes containing from 0.1 to 1 ug of NotI digested plasmid DNA.
5. Tubes are vortexed to mix the contents thoroughly, then incubated at 45^o for 30 minutes.
6. Cells are pelleted and resuspended in 400 ul of SC-leu. 200 ul is plated onto SC-leu medium. Plates are incubated at 30^oC for 3 to 4 days.
   

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