Isolate Plasmid DNA from Bacterial Cells

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BACKGROUND

A plasmid is a small DNA molecule inside a cell that is isolated from a chromosomal DNA and can replicate independently.¹ Most commonly these are found as small (usually 1-500 kb in size) circular, double stranded DNA molecules in bacteria; however plasmids are also present in eukaryotic organisms. Plasmid is replicon that is inherited in an extra chromosomal state.² Plasmids is considered as transferrable genetic elements or replicons.³ They are basically naked DNA. Plasmids are important mechanism in biotech labs where they are used to multiply particular genes.⁴ Plasmids carry genes that may serve survival of the organism by growing antibiotic resistance gene(R Plasmid) and production of restriction enzymes holds genes for utilization of some unusual metabolites, toxin production, nitrogen fixation, conjugation and some have no apparent function.⁵ The plasmids use the enzymes and proteins for replication encodedfrom the host chromosomal DNA.⁶

So the basic objective is isolation of plasmid DNA from bacterial cells.

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REQUIREMENTS

Media:             50 ml LB broth medium containing ampicillin as antibiotic

Equipments:    Micropipette

          Inoculating loops

          Sterile tips

          Eppendorf tubes

          Centrifuge tubes

          Markers

Reagents:        Solution 1: alkaline lysis solution (pH 8) – containing 50 mM glucose + 25 mM Tris + 10mM EDTA⁹

Solution 2: detergent for lysis (1% SDS + 0.2 N NaOH)

Solution 3: to maintain pH balance (3 M potassium acetate + 5 M glacial acetic acid)

TE Buffer: (10 mM Tris HCL+ 1Mm EDTA)

Ice cold ethanol (95%)

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PROCEDURE

The basic feature of plasmid which is essential for a high quality vector is that the small size of the plasmid is required to transfer large sized exogenous DNA. The unique restriction sites in plasmid help to insert the foreign DNA into the plasmid.⁸ The plasmid also contains some selectable markers or the markers may be inserted in order to confirm the transformation of the exogenous gene. Plasmids serve as important tool in genetics and biotechnology labs, where they are commonly used to multiply or express particular genes. The first task of the genetic engineering of plasmid is that it needs to be isolated from the cell.⁹

In plasmid isolating experiment, the modified alkali lysis method of bimboim and doly is followed. Inoculation of a single colony of E. coli is done and supplied to a 50 ml LB broth containing ampicillin antibiotic. The strain is resistant to ampicillin. The culture is grown for overnight.¹⁰

Select an inoculation of bacterial colony and then supply to 50 ml of LB broth containing ampicillin antibiotic and incubate at 37^oC for overnight. Then keep the culture at room temperature. For harvesting the culture, transfer it to 4 different 12.5 ml centrifuge tubes and then centrifuge it at 8000rpm for 5 minutes. Collect the pellets and discard the supernatant. Place the pellets to 100µl of solution1 from all the centrifuge tubes and then transfer to an Eppendorf tube. Then keep it for 5min at room temperature. Then add 200 µl Solution to the Eppendorf tubes and then for 10min keep in ice cool solution. Then add 150 µl pre-cooled Solution 3 to the Eppendorf tube and keep for 5min at ice-cooled temperature. Then vortex the tubes to make a uniform mixture. Centrifuge it for 5 min at 14000 rpm. Collect the supernatant in another Eppendorf tube and discard the pellet. Add 2 volume of ice-cooled 95% ethanol to the supernatant and keep in room temperature for 10min.Centrifuge the Eppendorf tube at 14000rpm for 5 min and collect the pellet. Wash the pellet and dry it with 70% ethanol solution. Place the pellet with 50 µl TE buffer and store it at -20^oC for future use.

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CONCLUSION

The isolated plasmid DNA is thus tested by GEL Electrophoresis for comparison.        

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REFERENCES

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