Frozen competent E. coli cells 【Carnegie Institution of Washington】
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Inoculate a 5ml overnight of E.coli in LB+20 mM MgSO4.
Next morning,inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml overnight culture.Grow at room temp (23℃)with good aeration (250rpm)to an A600 of 0.4-0.6.Temp is important--see original ref.
Place cells 10 min on ice.Transfer to a sterile bottle and spin 3K,10',4℃.
Resuspend pellet in 80 ml cold TB (swirl cells in bottle).Leave 10’/ice.
Spin cells 3K,10',4℃.
Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO.Leave 10'/ice.
Dispense into 220μl and 525μl aliquots (in cold sterile tubes)and freeze in dry ice/EtOH bath.Store -70℃.Typically,competency about 5X106 cfu/ug DNA.Note,improves after freezing.Cells good for a year and counting.
To use:
To 50 or 100μl cells,add 5-50 ng DNA.Leave 30’ on ice.
Heat shock,45 sec at42℃ then chill cells on ice about 2’.
Spin down (15 sec,eppendorf fuge)and remove SN.(Removing the Manganese seems to boost efficiency about 10X)and resuspend cells in 200μl LB.
For a supercoiled plasmid,plate 1μl of cells.For a ligation,plate 20μl and the rest.
TB (transformation buffer: filter sterilize and store 4℃)
| Product | [stock] | [ ]final | volumes to make 100ml |
| Pipes-NaOH pH6.7 | 0.5M | 10 mM | 2 ml |
| CaCl2 | 0.5M | 15 mM | 3 ml |
| KCl | 2M | 0.25M | 12.5 ml (or 1.864g) |
| MnCl2 | 1M | 55 mM | 5.5ml (or 1.088g) add to 100ml with ddH2 O |
