BIOL 1107L
Polymerase Chain Reaction Products
Complete this assignment in tandem with the lab. Please read through all posted content on
eLC for further information.
1. Give a brief description of the overall experimental design you will use to identify the
primer set of your DNA sample. (1.5 pts)
2. Determine the volumes that will be required for the forward/reverse primer mix in a 25
μL reaction, the volume of DNA to add for the final amount of 25 nanograms/ μL in the
reaction, and the volume of nuclease free water to be added. (1 pt)
PCR Reaction
Component Stock Concentration Component Volume Component Final Concentration
GoTaq Master Mix
2X with 400 μM dNTPs, 3mM
MgCL, and Taq DNA
polymerase
12.5 μl 1X solution
Forward/Reverse Primer
Mix 10 pmol/μl ?? 1.6 pmol/μl
DNA template 5 ng/μl ?? 1 ng/1 μl
Nuclease Free Water Not Applicable ?? Not Applicable
25 μl total reaction
volume
3. Using the volume obtained in question 2 for the Forward/Reverse Primer Mix, calculate
what the concentration (in pmol/μL) of the stock primers would be if there are 15
picomols of primer in the final reaction volume of 25 μL? (1 pt)
4. Given that the stock GoTaq Master Mix contains 400 μM of each of the dNTPs (dATP,
dTTP, dCTP, and dGTP) and that 1 pmol/μL is equal to 1 μM, what is the final amount of
dTTP contained in the final reaction volume of 25 μL? (1 pt)
The table below shows three primers for PCR targeting specific DNA regions. For each primer set,
“region” refers to the numbered base pair areas of DNA (total of 48,502 linear bp) that are copied
using the primer target sequences.
Table 1 Order manifest detailing the regions that each primer set will amplify on Lambda DNA
Set (Amplified region of DNA bps) Forward Primer Sequence Reverse Sequence
1 (23,449-26,916) 5’-AGC GTA TTA GCG ACC CAT CGT CTT-3’ 5’-AAG GCA TTC CGA GCA GAT GGT-3’
