Extraction of Plasmid and Gel Electrophoresis
Experiment title: Extraction of Bacteria Plasmid DNA and Analysis of extracted DNA
Samples
Objectives: 1) To study and understand the steps for extract bacteria plasmid DNA. 2) To measure the concentration and purity of extracted DNA by using spectrometric method and agarose gel electrophoresis method. 3) Determine the size of extracted DNA by using agarose gel electrophoresis method.
Materials and Methods:
(Refer to UDEE2124 lab manual from page 7 to page 10)
Results:
(I) Spectrophotometric Determination of DNA A260 = 3.923 A280 = 3.923 Conversion factor = 50 Dilution factor = 50
Therefore, dsDNA concentration (µg/ml) = A260 x conversion factor x dilution factor = 3.923 x 50 x 50 = 9807.5 µg/ml A260 / A280 = 1 Extracted plasmid DNA A260 / A280 < 1.8, therefore the extracted plasmid DNA contains impurity.
(II) Agarose Gel Electrophoresis
Figure 1: Extracted Plasmid DNA run on 0.8% agarose gel electrophoresis. The ladder used was VC 1kb DNA Ladder.
Fragment in Line S2 | Size of fragment (bp) | Types of fragment | a | <10000 | Linear | b | 10000 | Linear | c | 3000 | Supercoiled |
Table 1: Sizes and types of fragment in Figure 1.
Discussion:
Plasmid can carry one or more antibiotic resistance gene which makes the bacteria resistance to specific antibiotic. The reason ampicillin was added to LB medium during preparation of bacterial culture is to make sure that the E.coli plasmid produce ampicillin resistance gene. This will makes the plasmid which contains desired gene more easily to be isolated from self-ligated plasmid.
The bacteria plasmid DNA that being extracted is from E. coli by applying miniprep method. Solution I, Solution II, Solution III, ethanol and TE buffer are miniprep reagent. The inoculums of E. coli was undergoes the first centrifugation under 12,000rpm to separate out the
Samples
Objectives: 1) To study and understand the steps for extract bacteria plasmid DNA. 2) To measure the concentration and purity of extracted DNA by using spectrometric method and agarose gel electrophoresis method. 3) Determine the size of extracted DNA by using agarose gel electrophoresis method.
Materials and Methods:
(Refer to UDEE2124 lab manual from page 7 to page 10)
Results:
(I) Spectrophotometric Determination of DNA A260 = 3.923 A280 = 3.923 Conversion factor = 50 Dilution factor = 50
Therefore, dsDNA concentration (µg/ml) = A260 x conversion factor x dilution factor = 3.923 x 50 x 50 = 9807.5 µg/ml A260 / A280 = 1 Extracted plasmid DNA A260 / A280 < 1.8, therefore the extracted plasmid DNA contains impurity.
(II) Agarose Gel Electrophoresis
Figure 1: Extracted Plasmid DNA run on 0.8% agarose gel electrophoresis. The ladder used was VC 1kb DNA Ladder.
Fragment in Line S2 | Size of fragment (bp) | Types of fragment | a | <10000 | Linear | b | 10000 | Linear | c | 3000 | Supercoiled |
Table 1: Sizes and types of fragment in Figure 1.
Discussion:
Plasmid can carry one or more antibiotic resistance gene which makes the bacteria resistance to specific antibiotic. The reason ampicillin was added to LB medium during preparation of bacterial culture is to make sure that the E.coli plasmid produce ampicillin resistance gene. This will makes the plasmid which contains desired gene more easily to be isolated from self-ligated plasmid.
The bacteria plasmid DNA that being extracted is from E. coli by applying miniprep method. Solution I, Solution II, Solution III, ethanol and TE buffer are miniprep reagent. The inoculums of E. coli was undergoes the first centrifugation under 12,000rpm to separate out the
References: Birnboim, H.C. & Doly, J S 1979, A rapid alkaline procedure for screening recombinant plasmid DNA. Nucleic Acids, 7th edn. McGraw Hill, US. UTAR 2013, Extraction of Bacteria Plasmid DNA, Kampar. QIAGEN, n.d., Lysis of bavterial cells for plsmid purification, qiagen.com, Available from: < http://www.qiagen.com/literature/qiagennews/0299/992lysis.pdf >. [2 March 2013]. How to ectract plasmid DNA from bacterial cells, revolution.caret.cam.ac.uk, Available from:< http://revolution.caret.cam.ac.uk/flash/DNA_extraction_slideshow.swf>. [2 March 2013]. Addgene n.d., Inoculating a Liquid Bacterial Culture, Plasmid Protocol, Available from:< https://www.addgene.org/plasmid_protocols/inoculate_bacterial_culture/>. [2 March 2013].