Skip to content

Prepare chemically competent E. coli cells with TSS

This protocol is taken from an open source protocol, which was based on this paper.

Goal

Make a lab stock of chemically competent E. coli cells for routine plasmid cloning. We typically use NEB-Stable cells (ecHP56) for cloning in our lab.

Materials

  • Transformation and Storage Solution (TSS)
Component Amount Alternate
1M MgCl2 300 μL 60 mg MgCl2 hexahydrate
PEG 3350 1 g
LB to 10 mL
pH to 6.7 with NaOH or HCl
  • Filter through 0.2µ filter.
  • Add 500 μL of DMSO (not sure that DMSO is compatible with our 0.2μ filters, but it is unlikely to be contaminated)
  • Make fresh and store on ice.

Procedure

  1. Day 0: Streak NEB-Stable cells (ecHP56) on LB plates without antibiotic and incubate at 37°C overnight.
  2. Day 1: Start a 1.5 mL overnight LB culture by inoculating a single colony, and grow overnight with shaking or rolling at 37°C. Prepare 100 mL of LB in a 500-mL flask and autoclave 20 minutes to sterilize. Store at 37 °C overnight.
  3. Day 2: In the morning, dilute 1 mL of the overnight culture into the prewarmed flask and incubate at 37 °C with shaking at ~250 rpm.
  4. After one hour, check the culture. Check again every 20 minutes thereafter until it becomes turbid. Measure the OD600 as soon as it becomes turbid. Aim for OD600 between 0.3 and 0.5.
  5. While waiting for OD600 to reach 0.3—0.5, prepare TSS and store on ice. Chill a clinical centrifuge to 4 °C. Store two 50-mL conical tubes and a 5-mL serological pipet on ice.
  6. In a separate bucket, prepare ~100 PCR tubes (at least 12 x 8-tube strips) in an ice-water bath.
  7. When culture hits OD600 between 0.3—0.5, rapidly chill the culture by placing it on ice for 10 min.
  8. Split cold culture into the prechilled conical tubes and pellet 10 minutes, 3,000g, 4°C. Pour off supernatant and carefully remove the rest with a P1000.
  9. Resuspend the pellet in 10 mL of ice-cold TSS by gently pipetting with the chilled 5-mL serological pipet.
  10. Dispense 100 μL aliquots into the pre-chilled PCR strip tubes, and place immediately in −80°C box.

Checking transformation efficiency

  1. Thaw a 100 μL aliquot of cells on ice and split into two Eppendorf tubes on ice.
  2. Mix 1 μL of NEB pUC19 transformation control (50 pg/μL) with one 50 μL aliquot of competent cells.
  3. Mix 1 μL of TE or water with the other 50 μL aliquot of competent cells.
  4. Incubate on ice for 5 minutes.
  5. Plate directly onto prewarmed LB + carbenicillin plates (I use one plate, divided in half, for both transformations). Incubate overnight at 37 °C.
  6. Verify there are no colonies in the no-plasmid control. Then count colonies (CFUs) and note the transformation efficiency below, with the date. Calculate efficiency in transformants per μg as (number of CFUs) x (20,000).

Results

Prep by Prep date CFUs Efficiency (transformants/μg pUC19)
jyelland 2023-11-13 126 2.5 x 10^6