Preparing chemically competent cells

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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.
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*2.5 mL DMSO
*2.5 mL DMSO
*LB to 50 mL
*LB to 50 mL
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 +
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 15:50, 9 July 2005

Preparation

  1. Grow a 5 - 25 mL culture to an OD600 of 0.2 - 0.5.
  2. Centrifuge for 10 minutes at 3000 rpm and 4oC.
  3. Remove supernatant and replace with 10% original volume TSS.
  4. Make 100 uL aliquots and store at − 80oC.

Use

  1. Thaw TSS cells on ice.
  2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
    1. 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.
  3. Let sit for 30 minutes on ice.
    1. Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate cells for 30 seconds at 42oC.
    1. Note: According to the original TSS paper and qualitative experience, this step is completely optional and may actually reduce transformation efficiency.
  5. Add 1 mL SOC (2XYT and LB are also suitable) at room temp.
  6. Incubate for 1 hour at 37oC.
    1. Note: Can also save some time here by reducing incubation to ~45 min.
  7. 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.

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