Exp Of Plasmid

Universiti Tunku Abdul Rahman (Kampar Campus) Faculty of Science, Engineering, and Technology Bachelor of Science (Hons) Biotechnology Year 2 Semester 1 UESB 2142 Laboratory 2A (II) Principles of Biotechnology Lecturer: Dr. Choo Quok Cheong Student’s Name: Cheah Hong Leong Student’s ID: 08AIB03788 Experiment No. 2 and 3 Title: Extraction of Bacterial Plasmid DNA and Analysis of Extracted DNA Samples Date: July 7, 2009

Title: Extraction of Bacterial Plasmid DNA and Analysis of Extracted DNA Samples

Objectives: –

To learn the procedures needed in extracting the bacterial plasmid DNA

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To determine the concentration of original DNA sample and purity of prepared DNA sample by using spectrophotometer

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To analyze the extracted DNA sample by gel electrophoresis

Methods and Materials: Refer to laboratory manual UESB 2142, Laboratory 2A, and page 18, 19, 20, 21, 13, and 14.

Results: Part I. Spectrophotometric Determination of OD Reading and calculation on concentration of original DNA: Absorbance reading at 260 nm, A260 = 1.163 A Absorbance reading at 280 nm, A280 = 0.632 A Calculation: A260/ A280 ratio = 1.163 A/0.632 A = 1.8402 Conversion factor = 50µg/ml Dilution factor = (10µl + 990µl)/10µl = 100 Concentration of original DNA sample, µg/ml = A260 x conversion factor x dilution factor = 1.163A x 50µg/ml x 100 = 5815µg/ml

Part II. Agarose gel electrophoresis: Extracted DNA in well

1 kb Blue DNA Ladder Marker

Smear

Numbers of bands observed = 3 Estimation of the fragment sizes: 3500bp, 6000bp, and 10000bp.

Discussion: The A260 to A280 ratio of OD reading obtained from the spectrophotometer can be used to determine the purity of DNA extracted. The DNA extracted can be contaminated by the proteins and RNA molecules that were not totally purified from the DNA solution. If the ratio is less than 1.8, then the DNA extracted might be contaminated by proteins. The ration obtained from the calculation above, 1.8402 indicated the purity DNA extracted in experiment 2. Therefore the DNA extracted can undergo agarose gel electrophoresis with reliable result.

However, the result obtained from the gel electrophoresis did not show clear discrete fragments of DNA, but smears, except for the blue DNA ladder marker. One of the possible causes of this result was the concentration of the DNA extracted in the experiment 2. From the calculation, the concentration of original DNA sample was 5815µg/ml, which was might be too concentrated for the gel electrophoresis. The most suitable concentration of DNA sample for gel electrophoresis is below 80µg/ml, but if the concentration is exceeds 200µg/ml, it will start to affect the movement of the DNA molecules across the agarose gel. If the concentration is exceeds 800µg/ml, the apparent size of the DAN fragments would be about two-fold of the original size, thus greatly affecting and decreasing the motility of the molecules in agarose gel since electrophoresis is based in the molecular sizes. Therefore, too high of concentration of DNA sample largely prevents the accuracy of the gel electrophoresis, producing wide and unclear band that were visualized to be a smear. However, there were three bands that still visualized before the smear formed. The three bands: 3500bp, 6000bp, and 10000bp. But, the actual sizes of the plasmid DNA cannot be determine as the value given were unreliable. Conclusion: For accurate and precise gel electrophoresis, the concentration of DNA sample should not be exceeding 80µg/ml. Dilution of solution before loading the DNA into the well of agarose gel is needed if the concentration of DAN sample is found to be too high. References: Brown, T. A. (2006). Gene Cloning & DNA Analysis: An Introduction, 5th ed., Oxford, U.K: Balckwell Publishing Ltd. Dale, J. W. & Schantz, M.V. (2007). From Genes to Genomes: Concepts and Applications of DNA Technology, 2nd ed., West Sussex, U.K: John Wiley & Sons Ltd. Quick-reference: Determining DNA Concentration & Purity. (2007, August 22). Retrieved July 17, 2009, from http://bitesizebio.com/2007/08/22/dna-concentrationpurity/ Determination of DNA concentration by Spectrophotometric Estimation. (n.d.). Retrieved July 17, 2009, from http://homepages.bw.edu/~mbumbuli/molbio/labs/dna/index.html

DNA

Concentration

Calculator.

(n.d.).

Retrieved

July

18,

2009,

from

http://www.pubquizhelp.com/other/dnacalculator.html Doggett, N. A., Smith, C. L., & Cantorl, C. R. (1992). The Effect of DNA Concentration on Mobility in Pulsed Field Gel Electrophoresis. Nucleic Acid Research, 20, 859-864. Retrieved July 18, 2009, from http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=312029&blobtype=pdf