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Explain about the"Lazarus Technique"?
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Answer / swapna
It was a technique to recover bacteria i.e E. coli clones
from dried agar plates.
The following is the procedure for Lazarus Technique.
For dried agar plates cut out a section of plate with dried
bacterial residue.
Put in a micro centrifuge tube containing TE [10mM Tris-HCl
(pH 8), 1mM EDTA] and 1% SDS
For agar stabs add TE plus SDS directly into the stab vial
using an appropriate volume.
Using pipette tip, break up the agar into small fragments,
then decant the contents into a micro centrifuge tube.
** Vortex suspension at high speed for 3-5 min
** Spin at high speed for 30 sec
** Remove supernatant liquid to a fresh tube
** Extract supernatant with an equal volume of
phenol:chloroform (1:1). Centrifuge 5 min at high speed and
remove aqueous phase.
** Extract with an equal volume of chloroform. Centrifuge as
above and remove aqueous phase.
** To the aqueous phase, add one-tenth volume 3M sodium
acetate (pH 5.5) and two volumes absolute ethanol.
** Recover precipitated material by centrifugation at high
speed for 10min.
** Discard supernatant liquid and dissolve pellet in 10ul
sterile water
** Use entire amount for transformation of bacteria.
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