Preparing chemically competent cells: Difference between revisions
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#Plate 200 µL onto plate with appropriate antibiotic. | #Plate 200 µL onto plate with appropriate antibiotic. | ||
==Buffers== | |||
[[TSS]] (50 mL) | [[TSS]] (50 mL) | ||
*5g PEG 8000 | *5g PEG 8000 |
Revision as of 14:28, 5 July 2005
Preparation
- Grow a 5 - 25 mL culture to an OD600 of 0.2 - 0.5.
- Centrifuge for 10 minutes at 3000 rpm and [math]\displaystyle{ 4^o }[/math]C.
- Remove supernatant and replace with 10% original volume TSS.
- Make 100 uL aliquots and store at [math]\displaystyle{ -80^o }[/math]C.
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 [math]\displaystyle{ 42^o }[/math]C.
- Add 1 mL SOC (2XYT is also suitable) at room temp.
- Incubate for 1 hour at [math]\displaystyle{ 37^o }[/math]C.
- Plate 200 µL onto plate with appropriate antibiotic.
Buffers
TSS (50 mL)
- 5g PEG 8000
- 1.5 mL 1M MgCl2
- 2.5 mL DMSO
- LB to 50 mL