Ex04_S11-Analysis-1

Ex04_S11-Analysis-1 - Group Members Maria Bosquez Parxann...

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Group Members: Maria Bosquez, Parxann Counts, Valerie Lopez, Natalie Myers Exercise 4a Date: 2/10/11 LI Christina Hazard Unique 48835 Day: TH Room: PAI 1.18 Lab Start Time 9:00 BIO206L Spring 2011 Exercise 4 Analysis To be completed as a group and turned in at the beginning of your next laboratory period. Include your “Data & Results”, sketches, acquired digital images, etc. as directed by your laboratory instructor. Show your work for all calculations and/or print your MS Excel data sheets. Be sure to include proper units where necessary. Adhere to University’s Honor Code and course policies. Balance between brevity and completeness. 1. Understanding the basics of the polymerase chain reaction. a. For one of the PCR samples prepared by your group, list the contents of each tube and describe the role of each of the components found in the PCR reactions. DNA template- allows DNA polymerase to copy the DNA, human DNA obtained from buccal sample primers and dH2O- provide the 3’-OH group to which polymerase can add first nucleoside triphosphate magnesium concentration- a required co-factor for DNAses, role in catalyzing polymerization reaction deoxynucleotides (dNTPs)- substrate, a form of the required supply of 4 nitrogenous bases of DNA taq DNA polymerase and buffer- replicates DNA template at high temperatures b. What was the final volume of the reaction mix in the tube? 20 final volume c. What was the final concentration of the 2X Taq Buffer? 1X Taq buffer containing MgCl2 at 1.5 mM d. What is the function of the Chelex beads during genomic DNA isolation? What is the effect of Chelex beads on the subsequent PCR reaction using the genomic DNA you isolated? The Chelex beads bind divalent cations. Since Mg is a required co-factor of DNAses, the decreased availability of divalent cations decreases DNAse activity. The Chelex beads prevent the DNAses from breaking down DNA during boiling or inhibiting PCR reactions. 2. The rate of polymerization of Taq polymerase is 1000 bp/min. Your supervisor has asked you to PCR amplify an interesting DNA sequence that is of normal GC content and 1.5 kb in length. a. What elongation/polymerization time would you use in your PCR program? 1 minute and 30 seconds (extension times of one minute per 1000 base pairs) b. Given the number of starting template DNA molecules of about 1000 copies, approximately how many cycles would be required to amplify this target sequence to over 1 million copies? 1,000^x = 1,000,000 so 2 cycles over 1 billion copies? 1,000^x = 1,000,000,000 so 3 cycles over 1 trillion copies? 1,000^x = 1,000,000,000,000 so 4 cycles
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Group Members: Maria Bosquez, Parxann Counts, Valerie Lopez, Natalie Myers Exercise 4a Date: 2/10/11 LI Christina Hazard Unique 48835 Day: TH Room: PAI 1.18 Lab Start Time 9:00 c. How long would this take if a program were used similar to the standard PCR program listed on pg 107? If each standard elongation cycle takes about 1 minute for a 1 kb product, it would take 2 minutes to make 1 million copies, 3 minutes
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Ex04_S11-Analysis-1 - Group Members Maria Bosquez Parxann...

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