Q1: You Are Testing a Series of PCR Reactions at Different Temperatures

27 points total Biol203 Fall 2013

Homework 8:

Due in your lab starting Nov 13th and will be grading by your Lab TA.

Using Figure 1-

Q1: You are testing a series of PCR reactions at different temperatures.

Calculate the Tm of oligo 1 assuming 2 degree Celsius per A or T residues and 4 degree Celsius per G or C residue____54___

a: At that temperature calculated in Q1. Using oligos: 1, 2, 3, and 4.

What size PCR products are expected to form after a PCR reaction with:

1 and 4__238bp___

2 and 4____none____

3 and 4____ none _____

b: Using an annealing temperature of 26 degrees Celsius and letting the annealing reaction go for a few hours (not much energy available at room temperature to drive annealing of oligos), what size PCR products are expected to form after a PCR reaction with:

1 and 4__238bp_____

2 and 4____ none ______

3 and 4__238bp ____

Using Figure 2-

Q2: Using oligo 1 and 4, what are the expected sizes of the PCR products at the

annealing temperature of oligo 1,when trying to amplify SNP1 and SNP2 in heterozygous or homozygous mutants?

1 and 4 (Homozygous-SNP1)______none ______

1 and 4 (Heterozygous-SNP1 238bp _____

1 and 4 (Homozygous-SNP2)______none _____

1 and 4 (Heterozygous-SNP2)_____ 238bp _____

(use info on oligo 2 from Figure 1)

2 and 4 (Homozygous-SNP1)___ 238bp _____

2 and 4 (Heterozygous-SNP1)___238bp ___

2 and 4 (Homozygous-SNP2)_____ none ______

2 and 4 (Heterozygous-SNP2)____ none ______

(use info on oligo 3 from Figure 1)

3 and 4 (Homozygous-SNP1)_____ none ______

3 and 4 (Heterozygous-SNP1)____ none ______

3 and 4 (Homozygous-SNP2)___ 238bp ______

3 and 4 (Heterozygous-SNP2)___238bp _____

Using Figure 3-

Q3: SNP3 and SNP4 each cause the generation of a BamHI sites.

If you PCR genomic DNA heterozygous for SNP3 with:

oligos, 1 and 4, then what will be the approximate sizes of the fragments after BamHI digestion?___19bp and 219bp and 238bp___

oligos 2 and 4, then what will be the approximate sizes of the fragments after BamHI digestion?___none_____ and why______oligo won’t anneal______

oligos, 3 and 4 then what will be the approximate sizes of the fragments after BamHI digestion?____ none____and why______oligo won’t anneal ______

If you PCR genomic DNA homozygous for SNP3 with oligos:

1 and 4, then what will be the approximate sizes of the fragments after BamHI digestion?____19bp and 219bp ______

If you PCR genomic DNA heterozygous for SNP4 with oligos:

1 and 4, then what will be the approximate sizes of the fragments after BamHI

digestion?___ 2x 119bp and 238bp ______

If you PCR genomic DNA homozygous for SNP4 with oligos:

1 and 4, then what will be the approximate sizes of the fragments after BamHI

digestion?___ 2x 119bp ______