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PREPARATION OF COMPETENT CELLS USING CALCIUM CHLORIDE
Introduction Competent cells are those that possess more
easily altered cell walls that DNA can be passed through easily. These cells readily
incorporate foreign DNA. In order to make bacteria take in the plasmid, they must first
be made "competent" to take up DNA. This is done by creating small holes in the bacterial
cells by suspending them in a solution with a high concentration of calcium.
Materials Required LB broth , Culture plates , 1 M CaCl2.2H2O
(ice cold) , MgCl2 CaCl2 solution( ice cold ), Shaking incubator , Centrifuge , Inoculation
loop , Microfuge tubes , Polypropylene tubes , Pipettes , Tips.
Procedure Take an inoculation loop, flame it till red
hot and allow to cool for sometime
Pick a single bacterial colony from a plate and Transfer the colony into 10 ml LB broth.
Incubate the culture overnight at 37°C in shaking incubator
Take 1000 microlitre Micropipette, insert a fresh micropipette tip
Transfer 1 ml of the Overnight culture to 100 ml LB broth and incubate for 3 hours at
37°C in shaking incubator Transfer the bacterial cells to sterile, ice-cold
50ml polypropylene tubes. Store the tubes on ice for 10 minutes.
Centrifuge the cells at 4100 rpm for 10 minutes at 4°C.
Decant the medium from the cell pellets. Add 30 ml of ice-cold MgCl2-CaCl2 solution
to the pellet. Resuspend the pellet by swirling or gentle
vortexing in 30 ml of ice-cold MgCl2-CaCl2 solution.
Centrifuge the cells at 4100 rpm for 10 minutes at 4°C
Decant the medium from the cell pellets. Take 1000 microlitre Micropipette, insert
a fresh micropipette tip Transfer 2 ml of ice-cold 0.1M CaCl2 solution
to the pellet. Resuspend the pellet by swirling or gentle
vortexing Take the micropipette and adjust the volume
of the micropipette to 100 microlitres. Aliquot 100 microlitres of cells into each
vial, kept in ice Results
Thus competent cells were prepared using Calcium chloride method .You can use the cells directly
for transformation or freeze at -70°C.