Ex.

No: Date:

ISOLATION OF PLASMID DNA BY ALKALINE LYSIS METHOD

AIM: To isolate plasmid DNA by Alkaline Lysis Method. PRINCIPLE: Bacterial cells are lysed under alkaline conditions to liberate the cells contents. The double stranded DNA is denatured, and then it is neutralized with potassium acetate. The chromosomal DNA cannot reassociate & so, it is precipitated along with the cell debris on centrifugation, whereas, the plasmid being circular, reanneals & remains in the supernatant. It is precipated with ethanol. MATERIALS REQUIRED: Bacterial cells with PUC19, Solution I, Solution II, Solution III, Eppendorf tubes, centrifuge tubes, LB Broth, pipettes. CHEMICALS:

Solution I : 50 mM glucose 10 ml 1M Tris-cl (pH 8.2) 100 ml 10 mM EDTA (pH 8.0) 50 ml

Solution II: 0.2 N NaOH 10 ml 1% SDS 10 ml

Solution III: 5 M potassium acetate (pH 4.8) 10 ml

LB Broth isoamyl acetate ethanol 3 M sodium acetate TE buffer For agarose gel analysis: Agarose solutions Ethidium bromide Electrophoresis buffer

PROCEDURE: LB Broth containing Ampicillin at the rate of 50 g/ml was inoculated with a single colony of E.coli harboring PUC19.

It was grown up to log phase.

1.5 ml of the culture was centrifuged at 12,000 rpm for 2 minutes at 40C in order to pellet the cells.

The pellet was dried and suspend in 100 l of ice cold solution I. The cells were dispersed by vortexing. 200 l of freshly prepared solution II was added and the solution was mixed by inverting.

The Eppendorf was kept in ice for 10 minutes. 150 l of ice cold solution III was added and mixed well. It is kept in ice for 5 minutes at 40C.

The supernatant was transferred into another Eppendorf to that equal volume of isoamyl acetate was added and mixed well.

It was centrifuged at 12,000 rpm at 40C for 2 minutes. The upper aqueous phase was transferred to another Eppendorf. To that 2 volumes of ethanol and 1/10th volume of 3 M sodium acetate was added. It was kept at -200C for 1 hr.

Centrifuge at 12,000 rpm for 5 minutes at 40C to pellet DNA. The supernatant was discarded and the pellet was allowed to dry. It was dissolved in 50 l of TE buffer and the plasmid was checked in 1% agarose gel.

RESULT:

DISCUSSION:

REVIEW QUESTIONS: 1) Draw the structure of PUC19.

2) Differentiate between chromosomal and extra chromosomal DNA.

3) How will you prepare 100 ml of 0.5 M EDTA (pH 8.0)?