Competent cells

This is the procedure to prepare chemically competent E. coli for about 20 transformations. Scale the protocol up or down according to the specific needs.

• If using strains without plasmids, let them grow at 37degC in non-selective LB.
• If using strains that already have a plasmid, let them grow at 37degC in selective LB.
• If using strains with a temperature-sensitive plasmid, let them grow at 30degC in selective LB.

• DAY 1
  • Inoculate the desired strain from glycerol stock into LB and let it grow overnight at 37degC, 220 rpm.
• DAY 2
  • Re-inoculate 250 ul of cells in 50 ml of sterile pre-warmed LB in a sterile flask.
  • Incubate the flask under the same conditions as before, until cells reach OD600=0.4-0.5 (it should take 3-4 hours for the fast-growing strains, e.g. MG1655, while it may take longer for other strains). When in a hurry, a weaker dilution (e.g. 1 ml of cells in 50 ml of LB) can also be performed to decrease the waiting time.
  • Pre-chill the centrifuge (in the cell culture room) at 4degC and put in ice Buffer 1, Buffer 2, an empty 50-ml tube and 20 empty 0.5-ml tubes.
  • When the required OD600 is reached, transfer the whole cell culture in the 50-ml tube and centrifuge at 4000 rpm 10 min.
  • Discard supernatant and resuspend the pellet with 30 ml of Buffer 1.
  • Centrifuge as before.
  • Discard supernatant and resuspend the pellet with 2 ml of Buffer 2.
  • Aliquot in the 0.5-ml tubes (in ice) and store the vials at -80degC or use immediately for transformation.

Buffer 1

• Its composition is MgCl2 80mM, CaCl2 20mM. Prepare 100 ml of buffer by mixing 8 ml of MgCl2 1M, 2 ml of CaCl2 1M and 90 ml of autoclaved deionized water (from the blue tap of the Millipore machine).
• Store in the fridge and use near the flame.

Buffer 2

• Its composition is CaCl2 100mM, glycerol 15%. Prepare 100 ml of buffer by mixing 1 ml of CaCl2 1M, 18.75 ml of glycerol 80%
• Store in the fridge and use near the flame.

Glycerol 80%

• Mix 80 ml of glycerol with 20 ml of deionized water (blue tap of the Millipore machine).
• Autoclave in a pyrex bottle.
• Store at room temperature and use near the flame.

MgCl2 1M

• Dissolve powder in approximately 80 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
• Bring to a volume of 100 ml.
• Autoclave in a pyrex bottle.
• Store in the fridge and use near the flame.

CaCl2 1M

• Dissolve powder in approximately 80 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
• Bring to a volume of 100 ml.
• Autoclave in a pyrex bottle.
• Store in the fridge and use near the flame.