Magni:Protocols/Competentcells

From OpenWetWare

Jump to: navigation, search

Home        Contact        Lab Members        Publications        Research        Internal        Protocols        Data       


Contents

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.
Personal tools