This document provides instructions for making competent cells for transformation. It describes two transformation buffers, TFB1 and TFB2, listing the components and concentrations. TFB1 is used to incubate cells on ice for 30 minutes after centrifugation. TFB2 is then used to gently resuspend the cells before aliquoting and freezing at -80°C for long-term storage. The document also outlines 12 steps for the competent cell preparation process from growing an overnight culture to freezing the competent cells.
This document provides instructions for making competent cells for transformation. It describes two transformation buffers, TFB1 and TFB2, listing the components and concentrations. TFB1 is used to incubate cells on ice for 30 minutes after centrifugation. TFB2 is then used to gently resuspend the cells before aliquoting and freezing at -80°C for long-term storage. The document also outlines 12 steps for the competent cell preparation process from growing an overnight culture to freezing the competent cells.
This document provides instructions for making competent cells for transformation. It describes two transformation buffers, TFB1 and TFB2, listing the components and concentrations. TFB1 is used to incubate cells on ice for 30 minutes after centrifugation. TFB2 is then used to gently resuspend the cells before aliquoting and freezing at -80°C for long-term storage. The document also outlines 12 steps for the competent cell preparation process from growing an overnight culture to freezing the competent cells.
This document provides instructions for making competent cells for transformation. It describes two transformation buffers, TFB1 and TFB2, listing the components and concentrations. TFB1 is used to incubate cells on ice for 30 minutes after centrifugation. TFB2 is then used to gently resuspend the cells before aliquoting and freezing at -80°C for long-term storage. The document also outlines 12 steps for the competent cell preparation process from growing an overnight culture to freezing the competent cells.
Transformation Buffer 1 (TFB1): all concentrations final
30 mM potassium acetate 50 mM manganese chloride 100 mM potassium chloride 10 mM calcium chloride 15% v/v glycerol
pH to 5.8 with diluted (0.2 M) acetic acid
**throw away if overshoot pH 5.8 filter sterilize & store @ 4˚C
Transformation Buffer 2 (TFB2): all concentrations final
10 mM sodium MOPS pH 7.0
75 mM calcium chloride 10 mM potassium chloride 15% v/v glycerol
pH to 7.0 with diluted (0.2 M) NaOH
**throw away if overshoot pH 7.0 filter sterilize & store @ 4˚C 1. Start overnight LB culture (2-5 mL) -make sure have 2-50mL LB flasks ready for tomorrow -sterile labeled 1.5 mL tubes in racks @ -80˚C 2. Add overnight culture to each LB flask 3. Shake @ 37˚C until O.D. @ 600 nm = 0.5-0.6 4. Transfer culture to pre-cooled 50 mL centrifuge tubes 5. Spin cells down in pre-cooled 50 mL centrifuge tubes at 2500 rpm/4˚C for 15 minutes 6. Remove supernatant 7. Add 12.5 mL TF1 Buffer to each tube -GENTLY resuspend the pellet using the pipet -incubate cells on ice for 30 minutes 8. Spin cells down at 2500 rpm/4˚C for 15 minutes 9. Remove the supernatant 10. VERY VERY GENTLY add 4 mL TF2 Buffer to each pellet. VERY VERY GENTLY resuspend cells. (always keep butt of tube close to ice) 11. Now aliquot cells in -80˚C pre-cooled 1.5 mL tubes 12. Store tubes @ -80˚C