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

INTRODUCTION

                 DNA is the blueprint of life,if one interested in tuning the properties of life,DNA is the ultimate goal.In bacterial DNA recombinant technology,we often encounter plasmid which is a circular DNA which can replicate independently.Plasmid can grant various desirable characteristics to target bacteria,eg:Antibiotic resistant as shown in MRSA.

                  A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

                 Plasmid is small(hundreds to thousand of Kilobase) compared with the DNA. Plasmid can be transferred interspecific o r intraspecific. Before we can utilized plasmid as vector for transferring gene, we need to purify and isolate the plasmid in order to give a better access to restriction enzyme. Protease is also use to remove protein fragment that will contaminate the sample.
To extract DNA from a mixture of protein ,lipid and carbohydrate,the procedure is crucial in the experiments. Preparation are sort by size, minipreparation are used in the process of molecular cloning to analyze bacterial clones and yield a small weight of DNA.

               DNA filtration is the first and the most important step in DNA recombinant technology as the undesirable effect will amplified as the DNA will replicate many times. Hence, protocol must be followed in order to produce a consistent result in the product.

Objective

To understand the proper method needed to extract DNA from bacteria.

Method:

1.  1ml of bacteria culture is pelleted by centrifugation at 6 000 x g for 2 min at room temperature. The supernatant is decanted completely.

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2.   100µl Buffer R1 is added to the pellet and the cells are resuspended completely by   pipetting up and  down.

3.    For Gram-negative bacteria strains, 10µl lysozyme is added into the cell suspension. For Gram-positive bacteria strains, 20µl lysozyme is added into the cell suspension. The cell suspension is mixed thoroughly and incubated at 37°C for 20 min.

4. The pellet digested cells is centrifuged at 10 000 x g for 3 min. The supernatant is decanted completely.

1    5.  The pellet is resuspended in 180µl of Buffer R2 and 20µl of Proteinase K is added. It is mixed    thoroughly and incubated at 65°C for 20 min with occasional mixing every 5min.

      6.  400µl of Buffer BG is added and mixed thoroughly by inverting tube several times until a homogeneous  solution is obtained. The sample is incubated for 10 min at 65°C. 

       7.  200µl of absolute ethanol is added. The sample is mixed immediately and thoroughly.

       8.  The sample is transferred into a column assembled in a clean collection tube and centrifuged at 10 000 x    g for 1 min. The flow through is discarded.

      9.  The column is washed with 750µl of Wash Bufferand centrifuged at 10 000 x g for 1 min. The flow through is discarded.

    

     10.  The column is centrifuged at 10 000 x g for 1 min to remove residual ethanol.

    

     11.   The column is placed into a clean microcentrifuge tube. 100µl of preheated Elution Buffer is added directly onto column membrane and is stood for 2 min. The column is centrifuged at 10 000 x g for 1 min to elute DNA.

     12.  The sample is placed into the spectrophotometer to measure the reading of absorbance at 260nm and 280nm.

Results

Gram	Type of bacteria	260	280	Ratio (260/280)
Gram positive	Lactobacillus fermentation 8312	0.087	0.044	1.98
	Lactobacillus breris	0.130	0.181	0.72
Gram negative	E.coli	0.095	0.065	1.45
	Salmonella sp	0.109	0.094	1.16

Discussion:

From the calculation of result that we got from the photo spectrometer , the ratio of OD260 to OD280 for  Lactobacillus fermentation was  1.98. The value was very close to 2.0 and it showed that higher purity of RNA and contained less contaminants in the solution. Besides, the ratio of OD260 to OD280 that we got for  Escherichia coli and Salmonella were 1.45 and 1.16 respectively.The values were less than 1.6 and it showed that the solution contained a lot of contaminants. Not only that, the ratio of OD260 to OD280 for Lactobacillus breris was 0.72 and it showed that the solution contained even more contaminants like proteins , phenols and other contaminants.

So, we can know that the best ratio of OD260 to OD280 is 1.6-1.9. If the ratio is less than 1.6, the solution is contained some contaminants. However,if the ratio is more than 1.9,which mean that the solution is contained a higher purity of RNA. If the solution is in the range of such ratio stated, the solution contained a higher purity of DNA.

Other than that, we have discussed that some purposes of using different reagents for this experiment. For example, the purpose of adding lysozyme is to damage the bacterial cell wall by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan. Besides, the purpose of adding proteinase K is to cleave adjacent to carboxyl group of aromatic and aliphatic acid, it also includes in comprising the peptide cross linking bridge of peptidoglycan layer of cell wall of bacteria. The purpose of adding ethanol is to remove the salt out from nucleid acid, so the DNA molecules can become entangled with each other. The purpose f adding buffer R1 is to provide an optimum condition for Hydrogen to function effectively; Adding R2 is to privide an optimum condition for proteinase K to function efficiently; Adding elution buffer is to dilute the mixture of the solution and lastly, adding buffer BG is to trap the nucleid acid.

 Belows are some reasons of getting a low yield of DNA plasmid:

1.  Certain types of protein present in the mixture of protein and DNA.  
2. Plasmid DNA could not digested well/ did not deposit into wall of gel.
3.   Plasmid is denatured or incomplete protein denaturation.
4.  Concentrations of different samples.
5.   Efficiency of buffer elution is low.
6.  Cell suspension is incompleted.
7.   RNA contamination.

Conclusion

As a conclusion, in this experiment, we did not get the ratio as in the required ratio. This is because there are some mistakes that happened during handling out this experiment. So, in order to obtain the best ratio of OD260 and OD280 which are between 1.7 to 1.9, we have to ensure that the samples are in the correct required concentrations. Besides that, we also have to ensure that there is no contamination such as protein in the sample. Otherwise, it will affect the reading of the spectrophotometer and it will cause the ratio obtain are not in the range between 1.7 to 1.9.