Preparing chemically competent cells: Difference between revisions
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==Using== | ==Using== | ||
#Thaw TSS cells on ice. | #Thaw TSS cells on ice. | ||
#Add DNA, pipette gently to mix. | #Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough). | ||
#Let sit 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. | ||
#Add 1 mL [[SOC]] ( | #Add 1 mL [[SOC]] ([[2XYT]] is also suitable) at room temp. | ||
#Incubate for 1 hour at <math>37^o</math>C. | #Incubate for 1 hour at <math>37^o</math>C. | ||
#Plate 200 µL onto appropriate | #Plate 200 µL onto plate with appropriate antibiotic. | ||
Revision as of 08:31, 8 June 2005
Making
- 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.
Using
- Thaw TSS cells on ice.
- Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
- 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.
TSS (50 mL)
- 5g PEG 8000
- 1.5 mL 1M MgCl2
- 2.5 mL DMSO
- LB to 50 mL