EGY48 chemically Competent Cells
1*100ul
EGY48 chemically Competent Cells Components
EGY48 Competent Cell 100μ/tube Storage at -80鈩冿紙3month锛?/p>
pGADT7 (control vector, 10 ng/μl) 10μl Storage at -80鈩冿紙12month锛?/p>
Carrier DNA (5 μg/μl) 100μl Storage at -20鈩冿紙12month锛?/p>
PEG/LiAC 5ml Storage at 4鈩冿紙12month锛?/p>
EGY48 chemically Competent Cells Description
EGY48 strain is a strain by Clontech developed LexA system yeast two hybrid experiments, MAT alpha type, protein interaction can be verified or directly into plasmid library screening test; Transformation marker: his3, TRP1, URA3, LEU2; report gene as reporter gene UAS (upstream activation sequence from LexA op (x6)), only when the Bait and Prey interaction to start LEU2 expression. The EGY48-LexA yeast two heterozygous system needs three kinds of plasmids: pLexA, pB42AD and p8op-LacZ. Screening marker plasmid pLexA HIS3 for expression of DNA-BD (from prokaryotic consisting of 202 amino acid residues of LexA protein) and target protein (Bait) fusion protein; screening marker plasmid pB42AD TRP1 for expression of AD (from the herpes simplex virus 88 amino acids B42AD and target protein) protein (Prey) fusion protein; screening marker Report plasmid p8op-LacZ URA3, reporter gene LacZ, reporter gene UAS from LexA op (x6), only when the Bait and Prey interaction to start LacZ expression. The EGY48 receptive cells were made by special process, and could be stored at -80鈩?for three months. The pGBKT7 plasmid was used to detect the conversion efficiency of >104 cfu/ mu g DNA.
EGY48 chemically Competent Cells Protocol
1.Please store EGY48 chemically Competent Cells in -80°C for late use.
2. Thaw a tube of EGY48 chemically Competent Cells锛?00μl 锛塷n ice.
3. Add 2-5µl plasmid DNA, 10µl Carrier DNA(Heat shock at 95-100°C for 5min, then ice water imediately. Twice)锛?PEG/LiAc. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.
4. Place the mixture in the water at 30°C for 30 minutes, Flip 6-8times at 15min, Mixed uniformity.
5. Pellet the mixture by centrifugation at 500rpm/min for 40s. Pour off cell supernatant, then resuspend the cell pellet in the ddH2O 400ul.
6. Pellet the mixture by centrifugation for 30sec., Pour off cell supernatant. resuspend the cell pellet in the ddH2O 400ul.
7. Resuspend the cell pellet in the ddH2O 50ul.Spread the cell on the agar plates, then place it on 29°C for 48-96hr.