High Efficient K.Marxianus Transformation

  • At OD = 1.6
    1. Centrifuge at 2500g and resuspend in Competent buffer
    2. Incubate in Competent Buffer for 30min at RT
    3. Centrifuge at 2500g and resuspend in 1M Sorbitol (ice-cold)
    4. Centrifuge again and wash 3 times with 1M Sorbitol (ice-cold)
    5. After third wash take 1.5ml to microfuge tube and centrifuge at 2500g and resuspend pellet in 80ul (final volume)
# of Transformation1510
Volume of cells at OD=1.610ml50ml100ml
Competent Buffer8ml40ml80ml
Sorbitol1.5ml7.5ml15.5ml
3 x Sorbitol3×1.5ml3×7.5ml3×15.5ml
Final Sorbitol80μl400μl800μl
Add 1ml 1M sorbitol after elektro shock
Total Sorbitol7.08ml35.4ml70.8ml
1M Sorbitol182.17g
For 10ml1.82g
For 50ml9.1g
for 100ml18.2g
Competant Buffer8ml40ml80ml
100mM LiAc (1M)800μl4ml8ml
10mM DTT (1M)80μl400μl800μl
0.6M Sorbitol0.834g4.37g8.74g
10mM Tris-HCl pH 7.5 (0.5M)160μl800μl1.6ml