Mutagenizing a yeast gene with mTn-3xHA/lacZ 【Yale University】

Sequence of HAT tag (3xHA):

38 bp Terminal Repeat in upper case. loxR in bold.

GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGgc ggccattgaa ggtagaagag aaaatttgta cttccaaaga aagaaggccg ctatcgcttc ggataactcc tgctatacga agttatgggc ggccgtttac ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcc tatccatatg acgttccaga ttacgctccg gccgccCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCC

Bacterial strains used (provided in kit):

R1123	Strain XL1-blue carrying vector pHSS6.
R1236/B211	Strain RDP146 (F- recA' (delta lac-pro) rpsE; spectinomycin resistant) with plasmid pLB101 (pACYC184 with tnpA; active transposase, chloramphenicol resistant)(F. Heffron)
#95 /B427	Strain RDP146 with pOX38 F factor derivative carrying mTn3 derivative mTn-3xHA/lacZ (lacZ, URA3, tet; tetracycline resistant)
#70/B425	Strain NG135 (K12 recA56 gal-delS165 strA; streptomycin resistant) with plasmid pNG54 (pACYC184 with mTn3 res and tnpR seqs; active resolvase, chloramphenicol resistant)(N. Grindley)
B227	Strain DH5-alpha carrying pB227/GAL-cre (amp, ori, CEN, LEU2) (B. Sauer)

Vector used:

The accession for pHSS6 is M84115.

Antibiotics used:

Tetracycline, Tet (Sigma T3383)	12 mg/ ml in water. Use at 3 ug/ml (Tet3)
Kanamycin, Kan (Sigma K800)	10 mg/ ml in water. Use at 40 ug/ml (Kan40)
Chloramphenicol, Cm (Sigma C0378, I think)	34 mg/ml in ethanol. Use at 34 ug/ml (Cm34)
Streptomycin, Sm (Sigma S6501)	10 mg/ml in water. Use at 50 ug/ml (Sm50)
Ampicillin, Amp (Sigma A9518)	50 mg/ml in water. Use at 50 ug/ml (Amp50)

When only a few plates of each type are used, it's convenient to chop an LB plate up with a sterile toothpick, put the bits in a sterile flask, and melt the agar by microwave. Add appropriate amounts of antibiotic and repour plates.