BMBMethod:CompetentCells

From OpenWetWare
Jump to: navigation, search

<owwmenu font="arial, helvetica, sans-serif" bold="1" color="black" bgcolor="white" hovercolor="black" bghovercolor="gray" topFontSize="10" fontSize="8" pagewidth="650" image="OldMain2.jpg" lab="BMBMethod"> Home= Meetings=#,Schedule=Schedule, Past=Past, Presentations=Presentations Protocols=Protocols, Protein=Protocols#Protein, DNA=Protocols#DNA, RNA=Protocols#RNA, Other=Protocols#Other Tips & Tricks=TipsandTricks, Protein=TipsandTricks#Protein, DNA=TipsandTricks#DNA, RNA=TipsandTricks#RNA, Other=TipsandTricks#Other Equipment=Equipment, Departmental=Equipment#BMBDepartment, Personal=Equipment#Personal, Other=Equipment#Other Reagents=Reagents, Departmental=Reagents#Departmental, Cheap Buys=Reagents#Cheap Reagents </owwmenu>

Preparation of Chemically Competent Cells

Adapted from Inoue et al. 1990
Capable of ~3x109 cfu/ug

  1. Inoculate E. coli into 150mL SOB or LB media
  2. Grow between 18°C and 25°C until an OD600 of 0.5-0.6 is reached (overnight more convenient)
    1. At 18°C, it can certainly take 48+ hrs.
  3. Cool flask of cells on ice for 10 minutes
  4. Pellet cells by centrifugation at 4°C
  5. Gently resuspend pellet in 40mL of ice-cold TB
  6. Incubate cells on ice for 10 min
  7. Pellet cells by centrifugation at 4°C
  8. Resuspend cells in 5.58 mL of TB
  9. Add DMSO to final concentration of 7% (420 ul; 6mL final vol)
  10. Dispense into sterile microfuge tubes in 50-200ul aliquots
  11. Immediately snap freeze in liquid nitrogen
  12. Store in -80°C freezer until time to use


Transformation Buffer (TB) Recipe (50mL)

Reagent Amount Final Concentration
PIPES xxxg 10mM
CaCl2 xxxg 15mM
KCl xxxg 250mM
pH 6.7 (w/KOH) pH BEFORE adding MnCl2
MnCl2 xxxg 55mM












References

Inoue, H., Nojima, H., & Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. Gene, 96(1), 23–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/2265755