Exp. No. 8 Extraction and separation of Plasmid from Bacteria

 

Extraction and separation of Plasmid from Bacteria

Introduction

Plasmids are extra chromosomal genetic elements that permit microorganisms to store additional genetic information. This supplemental information is quite important for establishing mating capabilities, resistance to antibiotics and tolerance of toxic metals by microorganisms; but not all bacteria contain plasmids. Such additional genetic capability can permit the survival of the bacterium under conditions that are normally unfavorable for growth and survival.

Multiple drug resistance by the pathogenic bacteria is due to the presence of plasmids and this property of the bacteria has become a particular problem in treating some human infectious diseases because such bacteria are resistant to many antibiotics and can continue to grow in the body despite antibiotic treatment.

Plasmids are quite useful I research and are employed in genetic engineering as carriers of genetic information form a variety of sources. Plasmids are relatively small and are easy to manipulate. Plasmids can be isolated, genetic information from other sources can be spliced into them, and they can be implanted into viable bacterial cells, permitting expression of the genetic information they contain.

Plasmid isolation involves three steps:

1.      Growth of the bacteria and amplification of the plasmid

2.      Harvesting and lysis of the bacteria

3.      Purification of the plasmid DNA

The modification of the alkaline lysis method is followed for the plasmid isolation. Plasmid DNA is prepared from bacterial cells containing plasmid. The bacterial cells are lysed by treating the cells with a solution containing sodium dodecyl sulphate (SDS) and NaOH. SDS denatures bacterial proteins and NaOH denatures chromosomal and plasmid DNA. The mixtures is neutralized with potassium acetate, to make the plasmid DNA to reanneal rapidly. Most of the bacterial proteins and chromosomal DNA will be precipitated and forms a complex with potassium and are removed by centrifugation. The reannealed plasmid DNA is then concentrated by ethanol precipitation and in large preparation the plasmid is further purified by centrifugation to equilibrium in CsCl / ethidium bromide density gradients.

Aim

To isolate plasmid DNA from the given bacterial sample.

Procedure

·         Inoculate 5 ml of LB medium with a single bacterial colony. Grow the culture overnight.

·         Spin the culture in a centrifuge to pellet. Remove the supernatant using Pasteur pipette.

·         Suspend the pellet in 3.0 ml of TE buffer (pH – 8.0). 

·    Allow it to stand for 5 minutes at room temperature and make sure that all the cells are completely resuspended.

·         Add 2 volume of 0.2N NaOH and 1% SDS.

·      Mix by tapping the tube gently with finger and place on ice for 15 minutes. Note the mixture should become clear.

·         Add 1.5 ml volume of potassium acetate (3 M, pH 4.8) solution and vortex for 2 seconds to mix. Place on ice for 30 minutes to 1 hour.

·         Spin at 10,000 rpm for 15 minutes.

·         Remove the supernatant and add 4.5 ml of isopropyl ethanol (0.6 volume) or 2 volume of absolute alcohol.

·         Leave it for 30 minutes after gentle mixing.

·         Centrifuge at 10,000 rpm for 10 minutes and resuspend the pellet in TE buffer (50 µl).

·         Store the plasmid DNA at -20˚C.

The contaminating RNA can be destroyed by adding 1 µl of 10 µg/ml RNase solution. Most of the manipulation can be performed in microcentrifuge tubes.

Result

The sample should be observed in a UV visible spectrometer for its absorbance maxima. If the sample obtains a peak at the range of 260 nm then it is confirmed that the sample contains DNA in it.