Bio 142 - Week 5 slides - White sp2012

Bio 142 - Week 5 slides - White sp2012 - Gel...

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L. McLane, K. McClinic, T. Oliver and A. Escobar Gel Electrophoresis: Figuring out your repeat number and your ABO blood type!
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L. McLane, K. McClinic, T. Oliver and A. Escobar Before loading your gel, write down order in which your group will load your samples!! Add 2 to 2.5 μ L of 6x loading dye to the RFLP samples. Load pMCT118 samples first, followed by DNA marker (“ladder”), and finally, your RFLP samples.
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L. McLane, K. McClinic, T. Oliver and A. Escobar DNA - (BLACK) + (RED) RUN TO THE RED!
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L. McLane, K. McClinic, T. Oliver and A. Escobar Connect electrodes Run 60 - 75 minutes at 130V Take pictures using UV box Analysis
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Figures: Wheeler and Waikel Remember: Last week we performed a RESTRICTION DIGEST of your Exon 6 and 7 PCR products to figure out your blood type Total size of Exon 6 amplicon = 729 base pairs Total size of Exon 7 amplicon = 718 base pairs
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L. Mclane and A. Escobar “molecular scissors” bacterial enzymes that recognize specific base sequences Breaks the DNA sample into smaller pieces (hence, “fragment”) Digested DNA pieces can be separated by size on a gel. Restriction sites are typically 4-8 nucleotides in length and are often palindromic (read the same forwards and backwards). EcoR I - “sticky ends” http://en.wikipedia.org/wiki/Restriction_enzyme Sma I - “blunt ends” Review: Restriction Endonucleases
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Breaks the DNA sample into smaller pieces (hence, “fragment”), and then the pieces can be separated by size on a gel. Restriction enzymes are known to cut strands of DNA at specific regions and can be added to a DNA sample which has been subjected to the Polymerase Chain Reaction. http://www.scq.ubc.ca/wp-content/endonuclease2.gif Wheeler and Waikel R estriction F ragment L ength P olymorphism (RFLP)
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Restriction Enzyme Recognition Sites: Nar I: 5’…GG*CGCC…3’ 3’…CCGC*GG…5’ Pml I: 5’…CAC*GTG…3’ 3’…GTG*CAG…5’ Kpn I: 5’…GGTAC*C…3’ 3’…C*CATGG…5’ *Indicates Enzyme Cut Site The patterns of the bands that result from restriction digests in exons 6 and 7 will determine blood type. The digests will produce fragments of different sizes; the presence of these different bands will determine your blood type! RFLP Analysis of Blood Type Wheeler and Waikel
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L. McLane, K. McClinic, T. Oliver and A. Escobar Review for Exam I in 2 weeks (3/6 -3/9)
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L. McLane, K. McClinic, T. Oliver and A. Escobar
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L. McLane, K. McClinic, T. Oliver and A. Escobar Problem 1 1. You cannot answer A with certainty…in order to give a correct answer, you must look at more markers. This is first step to prove that he may be the father, but you can’t answer with certainty (see #3) Father B cannot be the father of the child (this you can answer with certainty because the child doesn’t have a marker that matches Father B) 2. Matches can easily occur at one loci, but matches at many loci would better indicate paternity
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L. McLane, K. McClinic, T. Oliver and A. Escobar
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L. McLane, K. McClinic, T. Oliver and A. Escobar
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This note was uploaded on 04/02/2012 for the course BIO 142L taught by Professor Conniearthur during the Spring '12 term at Emory.

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Bio 142 - Week 5 slides - White sp2012 - Gel...

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