LAB 6: Extraction of Plasmid DNA using GF-1 Plasmid DNA extraction Kit

INTRODUCTION

A plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).

Plasmid sizes vary from 1 to over 1,000 kilobase pairs (kbp). The number of identical plasmids in a single cell can range anywhere from one to even thousands under some circumstances. Plasmids can be considered part of the mobilome because they are often associated with conjugation, a mechanism of horizontal gene transfer.

Extraction is an easy and quick way to purify DNA from a mixture of proteins, lipids and nucleic acids (e.g., a cell or bacterial lysate). The mixture is extracted with phenol or a 50/50 mixture of phenol and chloroform. The organic solvents have two effects: 1.) they dissolve hydrophobic molecules and 2.) they denature proteins (which makes them insoluble in water). As a result, cell membranes and cellular proteins are either dissolved in the phenol/CHCl₃ (which is then discarded) or trapped in the interface between the two phases. DNA and RNA remain in the aqueous phase, and are easily separated.

The GF-1 Plasmid DNA Extraction Kit is designed for rapid and efficient purification of high copy and low copy plasmid DNA from bacterial lysates. This kit uses the alkaline lysis-SDS method to lyse cells and release plasmid DNA. Special buffers provided in the kit are optimized to enhance binding of DNA onto a specially-treated glass filter membrane for efficient recovery of highly pure plasmid DNA.

Among the features are it yields up to 20µg of DNA and multiple samples can be processed rapidly in less than 30 minutes.This process did not required organic-based extraction. It produced highly pure plasmid DNA ready to use for routine molecular biology applications such as restriction enzyme digestion, PCR, DNA sequencing, ligation, transformation and some mores.

Plasmid DNA Isolation Kits

RESULTS

Reading at 230 nm (OD230) = 0.241

Reading at 260 nm (OD260) = 0.389

Reading at 280 nm (OD280) = 0.195

To estimate the purity of the sample :

= Ratio of OD260/ OD280

= (0.389 ÷ 0.195)

= 1.995

To evaluate the level of salt carryover in the purified plasmid DNA :              

= Ratio of OD260/ OD230

= (0.389 ÷ 0.241)

= 1.614

DNA concentration (µg/mL)

= 50 µg/mL x OD260 x dilution factor

= 50 x 0.389 x 50

= 972.5 µg/mL

Total yield in 500µL sample

= DNA concentration x volume of sample in milliliters

= 972.5 µg/mL x 0.50 mL

= 486.3 µg

DISCUSSION

The quantity and purity of extracted plasmid DNA was determined. The mixture was transferred in a cuvette and the absorbance of the diluted sample was measured at 230nm, 260nm and OD280 nm in spectrophotometer.  The ratio between readings OD260 to OD280 that we got 1.995 closed to 1.8.  it shown that less contaminant with protein. If there is contamination with proteins, the OD260/OD280 will be significantly less.

Then, the level of salt carryover in the purified plasmid DNA was reading at 260nm and 230nm (OD260/OD230. The result showed (OD260/OD230) we got 1.614 it was greater than 1.5. From the reading it showed amount of our salt low because greater the amount of salt present, the lower ratio. DNA isolation are disruption of the cellular structure to create a lysate, separation of the soluble DNA from cell debris and other insoluble material and purification of the DNA of interest from soluble proteins and other nucleic acids. In the case of plasmid preparations, the multiple-day protocol typically involved cesium chloride banding followed by dialysis of the plasmid DNA.

The purified, high-quality DNA is ready to use for a wide variety of demanding downstream applications such as multiplex PCR, coupled in vitro transcription or translation systems, transfection and sequencing reactions. Number of methods has been developed to generate a cleared lysate that not only removes protein and lipids but also efficiently removes contaminating chromosomal DNA while leaving plasmid DNA free in solution. Methods for the preparation of cleared lysates that enrich for plasmid DNA include SDS-alkaline denaturation (Birnboim and Doly, 1979; Birnboim, 1983), salt-SDS precipitation (Hirt, 1967) and rapid boiling (Holmes and Quigley, 1981).

The SDS-alkaline denaturation method, which is used in all Promega plasmid isolation systems, is a popular procedure for purifying plasmid DNA because of its overall versatility and consistency. This technique exploits the difference in denaturation and renaturation characteristics of covalently closed circular plasmid DNA and chromosomal DNA fragments. Under alkaline conditions at pH 11, both plasmid and chromosomal DNA are efficiently denatured.

CONCLUSION

Extraction is an easy and quick way to purify DNA from a mixture of proteins, lipids and nucleic acids.

In a plasmid preparation it is always necessary to separate the plasmid DNA from the large amount of bacterial chromosomal DNA

Plasmids and the bacterial chromosome are circular, but during preparation of the cell extract the chromosome is always broken to give linear fragments. A method for separating circular from linear molecules will therefore result in pure plasmids.

REFERENCES

http://faculty.plattsburgh.edu/donald.slish/Extraction.html