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