Culturing Strains for Plasmid Isolation
MATERIALS REQUIRED:
1.LBA media - 100ml
2.Glycerol stock of DH5a
3.Ampicillin 50ul
4.Inoculation loop
5.Burner
6.Petri dish
...
INTRODUCTION: Characteristics of E.coli(DH5a):
It transforms with high efficiency. Like many cloning strains, DH5 alpha strain has several features that make it useful for recombinant DNA methods.
•The endA1 mutation inactivates an intracellular endonuclease that degrades plasmid DNA in many miniprep method.
•The hsdR17 mutation eliminates the restriction endonuclease of the EcoKI restriction-modification system, so DNA lacking the EcoKI methylation will not be degraded. DNA prepared from hsdR strains that are wt for hsdM will be methylated and can be used to transform with E. coli K-12 strains.
•M15 is the alpha acceptor allele needed for blue-white screening with many lacZ based vectors.
•recA eliminates homologous recombination. This makes the strain somewhat sickly, but reduces deletion formation and plasmid multimerization
PROCEDURE:
1.A glycerol stock of strain DH5a of E.coli was taken from -20.
2.It was thawed to bring down to room temperature.
3.A volume of 100ml of LBA media along with 50ul of ampicillin was plated into sterile petri dish.
4.From the stock a few cells were inoculated with the help of a loop into the plate.
PREPARATION OF PRIMARY CULTURE: We require 10ml of primary culture to prepare secondary culture, So to prepare 10ml primary culture, 5ml media was taken in 2 centrifuge tubes and 2 more tubes were prepared as a backup. So each centrifuge tube contains: 5ml media + 5ul of Ampicillin+1 healthy colony from the plate. It was incubated at 370 C at 180-200rpm for 14 – 16 hrs.
PREPARATION OF SECONDARY CULTURE:Each person require 1.5ml secondary culture. Hence a group would require, 1.5*5=7.5ml and 2.5ml as backup. So each centrifuge tube contains:10ml media+ 5ul of ampicillin+1ml of overnight primary culture.
Comment feed for this post
