Preparing chemically competent cells
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#Thaw TSS cells on ice. | #Thaw TSS cells on ice. | ||
#Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough). | #Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough). | ||
| + | ##Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help. | ||
#Let sit for 30 minutes on ice. | #Let sit for 30 minutes on ice. | ||
#Incubate cells for 30 seconds at <math>42^o</math>C. | #Incubate cells for 30 seconds at <math>42^o</math>C. | ||
Revision as of 11:22, 25 June 2005
Preparation
- Grow a 5 - 25 mL culture to an OD600 of 0.2 - 0.5.
- Centrifuge for 10 minutes at 3000 rpm and 4oC.
- Remove supernatant and replace with 10% original volume TSS.
- Make 100 uL aliquots and store at − 80oC.
Use
- Thaw TSS cells on ice.
- Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
- Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
- Let sit for 30 minutes on ice.
- Incubate cells for 30 seconds at 42oC.
- Add 1 mL SOC (2XYT is also suitable) at room temp.
- Incubate for 1 hour at 37oC.
- Plate 200 µL onto plate with appropriate antibiotic.
TSS (50 mL)
- 5g PEG 8000
- 1.5 mL 1M MgCl2
- 2.5 mL DMSO
- LB to 50 mL
