LAB 6: Bacterial DNA Extraction

INTRODUCTION

            Plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA which can replicate independently. Plasmid are commonly found in bacteria as small circular, double stranded DNA molecule.

            Plasmid are involved in the DNA recombinant experiment to produce large quantity of DNA. Plasmid act as vector to clone specific gene in DNA.  They are often found in Gram-positive bacteria and Gram-negative bacteria.    

            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. The 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 DNA onto a specially-treated glass filter membrane for efficient recovery of highly pure plasmid DNA. The kit will produce up to 20μg of DNA and produce the DNA rapidly. No organic-based extraction is needed. It also produce highly pure plasmid DNA which ready to use for routine molecular biology applications such as restriction enzyme digestion, PCR, ligation, DNA sequencing, transformation, etc.

            DNA purity can be determined by measuring the intensity of absorbance of the solution at wavelengths 260nm and 280nm.

OBJECTIVE

1.       To determine the method to extract the plasmid DNA from bacteria.

2.       To determine the difference of plasmid extraction between Gram-positive bacteria and Gram-negative bacteria.

MATERIALS AND REAGENTS

Centrifuge

Pipette

Incubator (Digital Dry Bath)

Column

Microcentrifugation tube

Buffer R1

Buffer R2

Proteinase K

Ethanol

Buffer BG

Wash Buffer

Elution Buffer

PROCEDURE

Reminder

·       All steps are to be carried out at room temperature unless stated otherwise.

·       Wash buffer (concentrate) has to be diluted with absolute ethanol before use.

·       Water bath was pre-set to 37°C and the second water bath to 65°C.

1. Centrifugation.

Pellet 1 ml of bacteria culture grown overnight or culture grown to log phase was centrifuged at 6,000 x g for 2 min at room temperature. The supernatant was decanted completely.

2. Re-suspension of pellet.

100µl Buffer R1 was added to the pellet and the cells were re-suspended completely by pipetting up and down or shake vigorously.

3. Lysozyme treatment.

For Gram-negative bacteria strains, 10µl lysozyme (50mg/ml) was added into the cell suspension. For Gram-positive bacteria strains,20µl lysozyme (50mg/ml) was added into the cell suspension. The mixtures were then mixed thoroughly and incubated at 37°C for 20 min.

4. Centrifugation.

Pellet was formed when digested cells were centrifuged at 10,000 x g for 3 min. The supernatant was decanted completely.

5. Protein denaturation.

Pellet was re-suspended in 180µl of Buffer R2 and 20µl of Proteinase K was added. The mixture was mixed thoroughly. They were then incubated at 65°C for 20 min in a shaking water bath or with occasional mixed every 5 min.

6. Homogenization.

2 volumes (~400µl without RNase A treatment, ~440µl with RNase A treatment) of Buffer BG was added and mixed thoroughly by inverting tube several times until a homogenous solution was obtained. It was incubated for 10 min at 65°C.

7. Addition of Ethanol.

200µl of absolute ethanol was added. The sample was mixed immediately and thoroughly.

8. Loading to column.

The sample (max. 650µl) was transferred into a column assembled in a clean collection tube (provided). It was then centrifuged at 10,000 x g for 1 min. Flow through was discarded. The remaining samples from step 7 were repeated the same step.

9. Column washing.

The column was washed with 650µl of Wash Buffer and centrifuged at 10,000 x g for 1 min. The flow through was discarded.

10. Column drying.

The column was centrifuged at 10,000 x g for 1 min. The residual ethanol was removed.

11. DNA elution.

The column was placed into a clean microcentrifugation tube. 50 µl of preheated Elution Buffer was added, TE buffer or sterile water directly onto column membrane and stand for 2 min. DNA was eluted by centrifuged it at 10,000 x g for 1 min. The DNA was then stored at 4°C or -20°C.

RESULT

	Gram Positive Bacteria	Gram Negative Bacteria
OD230	2.363	3.375
OD260	0.688	0.600
OD280	0.346	0.313
Ratio (OD260/OD280)	1.99	1.92
DNA Concentration (µg/mL)	674.509	588.240

DISCUSSION

1.       The bacterial has to be grown overnight to ensure that the bacterial is all grown to the phase needed.

2.     10µlof Lysozyme is added to the Gram Negative bacteria-strain while 20µl of lysozyme is added to the Gram Positive bacteria-strain because the Lysozyme is first needed to change the Gram Positive strain to Gram Negative and then deactivate both of it .

3.       During the extraction of DNA, there is a lot of contaminating proteins present. These contaminants must be removed. Proteinase K is used to digest these contaminating proteins. In addition, there may be enzymes that degrade nucleic acid present, proteinase K degrades these enzymes and protects the nucleic acids from attack. In addition, proteinase K is stable over a wide pH range and is well suited for use in DNA extraction.

4.       Ethanol is used in DNA extraction to force the DNA to precipitate in a solution. In order to collect a DNA sample, cells are broken down through agitation, then mixed with ethanol to create an aqueous solution. Ethanol works to prevent the DNA from dissolving into the water, instead causing it to precipitate out so it can be separated and extracted using a centrifuge.

5.       Ratio of OD260/OD280 is to to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. In this case, the ratio of both Gram Positive and Gram Negative Bacteria show ratio of 1.99 and 1.92 respectively indicates that the it shows the pure DNA.

6.       The DNA concentration is calculated by using Beer Lambert’s equation which is

            AxBxC whereby

            A: the wavelength-dependent absorptivity coefficient of OD260

            B: path length = 0.051 ml/µm

            C: analyte concentration = 0.02

CONCLUSION

Extraction of DNA is now becoming very important in research field as more product is coming out from the DNA especially human DNA by replicating it. In this case a pure and clean DNA has to be produce. And thus, the precautions have to be take noted and contamination of DNA must be avoided in order to produce pure DNA. The method of isolation of plasmid DNA can successfully extract the DNA out. The concentration of DNA extracted from gram positive bacteria is higher than the one from gram negative bacteria because the alkaline lysis is more effective towards the gram positive bacteria.