The paper “ Cleaning the Thermostable DNA Polymerase from Taq Polymerase Using the Latter to Enhance the Target Gene to Match the Unknown Entity” is an informative variant of a lab report on biology. The aim of this practical was to express and purify the thermostable DNA polymerase from Thermophilus aquatics using this Taq polymerase to amplify the target gene to identify the unknown organism. The polymerase chain reaction (PCR) is a tool that is used to amplify DNA, where at every thermal cycle a complementary DNA strand is synthesized. (Mullis et al 1986) With the repeated cycles of complementary DNA strand synthesis, there is an accumulation of newly synthesized DNA strands that could be modeled either to rapidly synthesis many strands of DNA or synthesis a few strands.
(Ruijter et al 2009, Rutledge and Stewart 2010) The PCR kit is a widely used technique in the amplification of DNA strands and as a result, it has many applications in various of synthesizing complementary DNA strands to make it possible to amplify a minutes DNA sample so that it could be detected if when it is a very small amount of DNA in any given sample.
The Taq polymerase first isolated from Thermophilus aquaticus is preferred because it could withstand high temperatures used in the PCR amplification of DNA strands. The PCR amplification of DNA strands takes place in several cycles of heating and cooling the thermal cycles stimulate the Taq polymerase primers to bind to the complementary DNA strands of the newly amplified DNA strands. The amplified DNA strands are then run in an SDS PAGE gel electrophoresis where the strands of DNA are separated according to there molecular weight, these separated DNA strands could then be visualized directly by staining with ethidium bromide.
(Erlich 1989)Techniques involvedTransformation and analysis of transformation efficiencyThe process of transformation bacterial cells to absorb DNA strands of another organism, the process to be used in this experiment is the chemical transformation process. Whereby the bacterial cells were treated with divalent metal ions to make cells competent to absorb DNA. Expression and Purification of Taq PolymeraseThe Taq DNA polymerase is used in PCR as a primer to bind to the DNA in the sample so that the complementary synthesis of new DNA strands takes place.
(Engelke et al 1990) In the presence of a DNA template in the sample, the Taq polymerase primer terminus and Mg++ catalyzes the incorporation of dNTPs into sample DNA because the Taq polymerase contains a polymerization dependent 5'-3' exonuclease activity that initiated elongation process of DNA synthesis. (Grimm and Arbuthnot 1995)The expression of the Taq polymerase gene is under the control of the lac promoter system. This means that if the Taq polymerase gene has to be expressed galactose has to be present in media.
In this experiment, the non-hydrolyzable galactose analog IPTG was used to induce the cells to express a protein. The cells cannot metabolize IPTG, therefore, the cells will continuously keep expressing the protein. (Saiki 1985)SDS-PolyAcrylamide Gel ElectrophoresisThe SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is used to separate DNA according to its molecular weight. The DNA double-strand is separated into single strands in the SDS-loading buffer where the detergent Sodium Dodecyl Sulphate (SDS) binds to the single-stranded DNA, keeping it unfolded and giving it a uniform mass to charge ratio when the gel is run.
Using SDS-PAGE you will be able to show the DNA present in your sample. This will allow you to show the purification of the Taq polymerase through the steps of expression and purification. You will need to cast an SDS-PAGE gel. Each gel will allow you to run a total of 10 samples so the gel can be shared between two. (Grimm and Arbuthnot 1995) Used a prepared Taq polymerase from fellow bench members and the provided Taq polymerase by staff members and the samples were run in the thermal cycler by the staff.
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