LAB 6: EXTRACTION OF PLASMID DNA USING GF-1 PLASMID DNA EXTRACTION KIT

INTRODUCTION

 The  GF-1 Plasmid DNA Extraction Kit is designed for rapid and efficient purification of

high copy and low copy plasmid DNA without the need for precipitation or organic

extractions. This it uses a specially-treated glass filter membrane fixed into a column to

efficiently bind DNA in the presence of high salt. Combining alkaline lysis-SDS and minicolumn spin technology, up o 20μg of plasmid DNA from bacterial cultures can be isolated. Multiple samples can be processed rapidly and with practice, the purification takes less than 30 minutes. Optimized buffers ensure only highly pure plasmid DNA is extracted and is ready to use in many routine molecular biology applications such as restriction enzyme digestion, radioactive/fluorescence DNA sequencing, PCR, ligation, transformation and other

manipulations.

OBJECTIVE:
to extract plasmid DNA from plasmid-containing culture.

DISCUSSION :

Before the extraction started,dilute concentrate Wash buffer and washing steps are necessary to remove unbound reagents and reduce background, thereby increasing the signal:noise ratio. Insufficient washing will allow high background, while excessive washing may result in decreased sensitivity caused by elution of the antibody and/or antigen from the blot. Occasionally, wash buffer formulations consist of only a physiological buffer such as Tris buffered saline (TBS) or phosphate buffered saline (PBS) without any additives. More commonly, a detergent such as 0.05% Tween 20 is added to the buffer to help remove nonspecifically bound material. Depending on the specifics of the assay, the amount of detergent in the wash buffer will vary, though typical concentrations are from 0.05 to 0.5% for detergents like Tween-20*. Another common technique is to add a 1:10 dilution of the blocking solution to the wash buffer. Including the blocking agent with the detergent may help to minimize background in the assay by preventing elution of the blocking protein from the membrane and/or allowing nonspecific interactions to occur with the protein in solution rather than those immobilized on the membrane.

Ratio between the readings (OD260/OD280) provides an estimate of the purify of the sample. Pure plasmid DNA has ratio 1.8691. If there is contamination proteins,the OD260/OD280 will be significantly less. Meanwhile the ratio between the readings at (OD260/OD230) evaluates the level of salt carryover in the plasmid DNA.The OD260/OD230 is best if greater than 1.5 and the result is 1.6376.Lower the ratio if greater the amount of salt that is present. 

 In a spectrophotometer, a sample is exposed to ultraviolet light at 260 nm, and a photo-detector measures the light that passes through the sample. Using the Beer Lambert Law it is possible to relate the amount of light absorbed to the concentration of the absorbing molecule. At a wavelength of 260 nm, the average extinction coefficient for double-stranded DNA is 0.020 (μg/ml)^-1 cm^-1, for single-stranded DNA and RNA it is 0.027 (μg/ml)^-1 cm^-1 and for short single-stranded oligonucleotides it is dependent on the length and base composition. Thus, an optical density (or "OD") of 1 corresponds to a concentration of 50 μg/ml for double-stranded DNA. This method of calculation is valid for up to an OD of at least 2.

To determine the quantity and purity of extracted plasmid DNA,there are some step of calculation required.

DNA concentration (µg/mL):

= 50 µg/mL x OD260 x dilution factor

Dilution factor is the final volume / aliquot volume.aliquot volume is the measure of sub volume of original sample . final volume is the total volume.

dilution factor =final volume /aliquot vol.

The total yield in 500µL sample:

= DNA concentration x volume of sample in milliliters

The concentration of the plasmid is dependent on copy number and elution volume. If a higher concentration is desired for subsequent applications, perform an ethanol precipitation after plasmid isolation.

 RESULTS:

Photometry reading:

OD230 = 0.218

OD260 = 0.357

OD280 = 0.191

Ratio of (OD260/ OD280):

= (0.357 ÷ 0.191)

= 1.8691

Ratio of (OD260/ OD230):

= (0.357 ÷ 0.218)

= 1.6376

DNA concentration (µg/mL):

= 50 µg/mL x OD260 x dilution factor

= 50 x 0.357 x 50

= 892.5 µg/mL

Total yield in 500µL sample:

= DNA concentration x volume of sample in milliliters

= 892.5 µg/mL x 0.50 mL

= 446.25 µg

CONCLUSION:

 As a conclusion, with advances in the development of DNA vaccines and gene therapy, there is a growing need for plasmid DNA with high quality for fundamental research and clinical trials. During this experiment the student carry out the extraction of the plasmid DNA from plasmid-containing culture. This process is based on alkaline lysis and can be easily scaled up to meet demands for larger quantities. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to affect lysis and control alkalinity.