Preparing chemically competent cells: Difference between revisions
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==Making== | |||
#Grow a 5 - 25 mL culture to an OD<sub>600</sub> of 0.2 - 0.5. | |||
#Centrifuge for 10 minutes at 3000 rpm and <math>4^o</math>C. | |||
#Remove supernatant and replace with 10% original volume [[TSS]]. | |||
#Make 100 uL aliquots and store at <math>-80^o</math>C. | |||
==Using== | |||
#Thaw TSS cells on ice. | |||
#Add DNA, pipette gently to mix. | |||
#Let sit 30 minutes on ice. | |||
#Incubate cells for 30 seconds at <math>42^o</math>C. | |||
#Add 1 mL [[SOC]] (Room Temp). | |||
#Incubate for 1 hour at <math>37^o</math>C. | |||
#Plate 200 µL onto appropriate resistance plate. | |||
Revision as of 19:24, 6 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.
- Let sit 30 minutes on ice.
- Incubate cells for 30 seconds at [math]\displaystyle{ 42^o }[/math]C.
- Add 1 mL SOC (Room Temp).
- Incubate for 1 hour at [math]\displaystyle{ 37^o }[/math]C.
- Plate 200 µL onto appropriate resistance plate.
TSS (50 mL)
- 5g PEG 8000
- 1.5 mL 1M MgCl2
- 2.5 mL DMSO
- LB to 50 mL
