2010 Bio 311 Lab C Manual (part 1)

2010 Bio 311 Lab C Manual (part 1) - Figure by Dr George...

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76 http://web.indstate.edu/thcme/mwking/pcr.gif Figure by Dr. George Pearson, Oregon State University taken from www.orst.edu/instruction/bb331/ lecture03/FigI.html ,
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77 C1. . Lab Overview: You will resuspend colonies from your experimental plate in water. You will use part of the resuspended cells to grow bacteria for next week lab and the other part to make crude plasmid DNA by disrupting the cells with heat. For this lab, the crude plasmid DNA will be used for polymerase chain. A section of the plasmid DNA in the lysates will be amplified by using primers that flank the polylinker of the pET28b vector. You will view your PCR products (amplicons) on an agarose gel during lab C2. Background Polymerase Chain Reaction (PCR) is a powerful tool for making many copies of DNA. DNA taken from a single cell (hair follicle, sperm, blood, etc.) can be amplified and then analyzed. You can clone a specific gene from that cell within hours rather than days. This is the basis of many Forensic DNA technologies. PCR requires several components that make up a master mix, to work: Taq Polymerase : an enzyme that makes copies of DNA from DNA templates. It is heat stable, and therefore remains active during the high temperatures necessary to complete the PCR cycles. Template DNA: the DNA that the Polymerase copies from. Buffer : to ensure a stable environment for the enzyme that copies the DNA. MgCl 2 : a cofactor required by Polymerase dNTPs : nucleotide building blocks that Polymerase strings together to make the new DNA strands. Primers : short, pieces of single-stranded DNA that hybridize or anneal (complementary base pair) near the region of DNA that you wish to amplify. DNA Polymerases can only extend a pre-existing DNA chain. Therefore, Polymerase begins synthesis starting from the 3' ends of the primers that have hybridized to the template DNA. A pair of primers is required. The two primers hybridize to opposite strands of the DNA so that their 3’ ends face each other. Once all these reagents are added to the reaction tube, the tube is placed in the thermal cycler and the contents are cycled through 3 steps: a denaturation step , annealing step , and elongation step Before Polymerase can copy either strand of DNA, the DNA strands must be separated from each other or denatured. This occurs when DNA is heated to temperatures around 94 0 C. Once the template DNA is single-stranded, then the temperature is lowered to the annealing temperature for the annealing step. The primers anneal to the melted template following base pair rules: A - T and G - C. The optimal annealing temperature must be empirically determined for each ± Read "An Overview of the BCE labs" and Lab ± State lab Goal(s) ± Outline or make a flow chart of the methods.
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2010 Bio 311 Lab C Manual (part 1) - Figure by Dr George...

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