AH109 chemically Competent Cells
Genotype
MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, MEL1 GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ
Components in AH109 chemically Competent Cells
AH109 Competent Cell: 100μl/tube Storage at -80℃（3month）
pGADT7 (control vector, 10 ng/μl) 10μl Storage at -80℃（12month）
Carrier DNA (5 μg/μl) 100μl Storage at -20℃（12month）
PEG/LiAC: 5ml Storage at 4℃（12month）
AH109 chemically Competent Cells Operation
1.Please store AH109 chemically Competent Cells in -80°C for late use.
2. Thaw a tube of AH109 chemically Competent Cells（100μl ）on ice.
3. Add 2-5µl plasmid DNA, 10µl Carrier DNA(Heat shock at 95-100°C for 5min, then ice water imediately. Twice)， 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.
Media preparation
1) YPDA (1L):
Tryptone 20g
Yeast extract 10g
0.2% adenine 15ml
Add Water to 950ml, PH to 6.5 with hydrochloric acid;
Agar 20g (for plates only)
15 min high pressure sterilization at 121 centigrade,
When the temperature of the medium is reduced to 55 degrees C, the filter has been filtered.
40% glucose and 50 ml
2) SD medium (1L):
Yeast Nitrogen base 6.7g
Glucose 20g
Dropout (according to the instructions)
Add water to 1L, adjust PH to 5.8;
Agar 20g (for plates only)
15 min high pressure sterilization at 121 centigrade,
3) 0.2% adenine (1L)
Adenine 2g
Add water to to 1L; dissolved after autoclaving or 0.22 m membrane filtration.
