Group Members: Maria Bosquez, Parxann Counts, Valerie Lopez, Natalie Myers
LI Christina Hazard
Room: PAI 1.18
Lab Start Time 9:00
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
Show your work for all calculations and/or print your MS Excel
Be sure to include
proper units where necessary.
Adhere to University’s Honor Code and course policies.
between brevity and completeness.
Understanding the basics of the polymerase chain reaction.
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
magnesium concentration- a required co-factor for DNAses, role in catalyzing
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
What was the final volume of the reaction mix in the tube?
What was the final concentration of the 2X Taq Buffer?
1X Taq buffer containing MgCl2 at 1.5 mM
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
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.
The rate of polymerization of
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.
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)
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