Bacterial Transformation - Hanahan Method
Jeff Lawrence
1. Inoculate 1 mL SOB with a single colony. Vortex and use to innoculate 50 mL SOB. Grow to Klett 140, or OD 0.5 @ 550 nm (5x108 cells/mL). SOB is :
2.0 % Typtone 20 g Bactotyptone
0.5 % Yeast Extract 5 g Yeast Extract
10.0 mM NaCl 2.0 mL 5 M NaCl
2.5 mM KCl 2.5 mL 1 M KCl
10.0 mM MgCl2 10.0 mL 1 M MgCl2
10.0 mM MgSO4 10.0 mL 1 M MgSO4
930 mL ddH2O
2. Chill on ice 15 min. Centrifuge at 4000 g for 12 min at 4°. Resuspend in 16 mL cold FSB:
10 mM KOAc 10 mL 1 M KOAc, pH 7.0
100 mM KCl 100 mL 1 M KCl
45 mM MnCl2 45 mL 1 M MnCl2
10 mM CaCl2 10 mL 1 M CaCl2
3 mM HACoCl2 30 mL 100 mM HACoCl2
10 % Glycerol 200 mL 50% Glycerol
605 mL ddH2O
3. Chill on ice 15 min. Centrifuge at 4000 g for 12 min at 4°. Resuspend in 4 mL cold FSB.
4. Add 140 µL fresh DMSO. Chill on ice for 5 min.
5. Add 150 µL of DTT Solution:
2.25 M DTT 900 µL 2.5 M DTT
40.0 mM KOAc, pH 6.0 40 µL 1 M KOAc, pH 6.0
60 µL ddH2O
6. Chill on ice 10 min. Add 140 µL fresh DMSO and incubate on ice 5 min.
7. Aliquot 200 µL cells into chilled 5 mL tubes. Add up to 100 ng DNA in less than 10 µL TE. Swirl and incubate on ice for 30 min.
8. Place samples at 42° for 90 sec. Rechill on ice for 2 min.
9. Add 800 µL SOC:
1 X SOB 100 mL SOB
20 mM Glucose 200 mL 1 M Glucose
10. Incubate at 37° for 60 min with swirling. Plate on selective media.
Last Update: Thursday June 19 2014
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Eric Kofoid
eckofoid at ucdavis.edu