1107LWk8RameshA

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1107L

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Biology

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Jan 9, 2024

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BIOL 1107L PCR Concentration & Products Complete this assignment in tandem with the lab. Please read through all posted content on eLC for further information. 1. What is the concentration (in picomol/μL) of Spike's stock primers? We know from our other lab technician, Hairy Dawg, that there are 20 picomols of primer in the final reaction volume. It might be helpful to review the PCR reaction content posted on eLC. (1.5 pts) 5pmol/ μL 2. How many nanograms of Lambda DNA will be in the PCR reaction? (1 pt) 25 ng The table below shows three primers for PCR targeting specific  DNA regions. For each primer set, “ region” refers to the numbered base pai r 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’ 2 (22,985-27,342) 5’-CTC TGT CAG CTG CAT AAC GCC AAA-3’ 5’-TGT TAT GAG GGA GCG GAA GTT-3’ 3 (35,454-40,049) 5’-AAT CGC ACT TAC GGC CAA TGC TTC-3’ 5’-CAG CCA GTA GTG GGC GTT TGA TTT-3’ Primer set U 3. Given the information above, calculate the length of the PCR product for each primer set in base pairs (bp). (1.5 pts) Unknown PCR Product (bp) Primer Set #1 3467 Primer Set #2 4357 Primer Set #3 4595 4. Describe how the following conditions would affect the PCR product and explain your reasoning. Use the PCR protocol posted in eLC under Molecular Applications – Using PCR. (6 pts)  Half the standard dNTP concentration in the master mix Half the standard dTNP would probably result in the DNA polymerase not having enough dNTPs to extend the primers for PCR.

BIOL 1107L PCR Concentration & Products  A denaturation temperature set at 60 °C With a temperature of 60°C, the temperature will be too low, which means that denaturation will not complete, and the amplication rate will be lower.  An extension time of 30 seconds 30 seconds will be too short of a time for taq polymerase to make the strand of DNA.  Programming only 10 cycles of PCR instead of the required 40 There will not be enough amplification products with only ten cycles of PCR.

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