Preparing chemically competent cells: Difference between revisions
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##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. | ##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. | ||
##Note: If you are in a rush, you can shorten this incubation time to 5-10 min. | |||
#Incubate cells for 30 seconds at <math>42^o</math>C. | #Incubate cells for 30 seconds at <math>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. | ##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. | #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. | ||
##Note: Can also save some time here by reducing incubation to ~45 min. | |||
#Plate 200 µL onto plate with appropriate antibiotic. | #Plate 200 µL onto plate with appropriate antibiotic. | ||
| Line 22: | Line 24: | ||
*2.5 mL DMSO | *2.5 mL DMSO | ||
*LB to 50 mL | *LB to 50 mL | ||
Filter sterilize (0.22 µm filter) and store at 4 ˚C or -20 ˚C. | |||
[[Category:E.Coli Protocol]] | [[Category:E.Coli Protocol]] | ||
Revision as of 12:50, 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.
- Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
- 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.
- Note: Can also save some time here by reducing incubation to ~45 min.
- 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
Filter sterilize (0.22 µm filter) and store at 4 ˚C or -20 ˚C.
