Preparing chemically competent cells: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Barry Canton (talk | contribs) No edit summary |
(→Use) |
||
Line 11: | Line 11: | ||
#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. | ||
#Add 1 mL [[SOC]] ([[2XYT]] | ##Note: According to the original TSS paper and qualitative experience, this step is completely optional and may actually reduce transformation efficiency. | ||
#Add 1 mL [[SOC]] ([[2XYT]] and LB are 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 plate with appropriate antibiotic. | #Plate 200 µL onto plate with appropriate antibiotic. |
Revision as of 12:28, 9 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.
- Note: According to the original TSS paper and qualitative experience, this step is completely optional and may actually reduce transformation efficiency.
- Add 1 mL SOC (2XYT and LB are 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