Metabolic Biochemistry Worksheet

Student Name: Jessica Sousa

Student Number: M00455072

Date: 15/11/2014

BMS2113 Molecular and Metabolic Biochemistry

Module Leader: BeataBurczynska
Practical Tutors: Dirk Wildeboer&BeataBurczynska

Formative practical worksheet (three questions)

Page limit: Cover sheet + 2 pages maximum

By completing the coversheet, students acknowledge the following on the originality of their work:
I confirm that this work is my own work; where I have rewritten someone else 's ideas in my own words, I have identified this with a reference; where I have used words directly from a book or any other source, I have enclosed these in quotation marks and have identified the source in a reference (although I am advised that the latter is not good practice).
RNase specifically attacks pyrimidine nucleotides by cleaving the phosphodiester bond 5 '-ribose of a nucleotide and the phosphate group attached to the 3 '-ribose of an adjacent pyrimidine nucleotide which results in 2 ', 3 '-cyclic phosphate is hydrolyzed to the corresponding 3 '-nucleoside phosphate (Blackburn and Moore, 1982).

c) 96% ethanol

Ethanol when added to the solution will cause the DNA to precipitate so that will become visible. Precipitation will separate the DNA from other cellular components which are soluble in alcohol. DNA will clump together and rise into the alcohol layer forming into a white stringy solid.

2. Calculate the following for your extracted DNA sample, showing each step of your calculations. Discuss concentration, yield and purity referring to what was expected or required for the experiment one sentence for each.

a) Concentration

A1 reading = 0.170 Abs at 260 nm
A2 reading = 0.069 Abs 280 nm
F (dilution factor) 100/10 = 10

DNA conc. (µg/mL) = A260 nm x F x 50 µg/mL = 0.170 x 10 x 50 = 85
The result obtained is too low and is not in the range expected. The low value of the DNA purity can be due to protein contamination, inadequate pipetting, RNA contamination, inadequate use of the centrifugue.

3. What was the PCR programme required for your PCR reaction in practical 1.Include all steps, temperatures and times. Briefly describe what was happening in each step and why you used certain temperatures.

The first step of the PCR reaction the thermocycler was programmed to 95ºC for the Denaturation of DNA template. In this step, the double-stranded DNA is separated into two single-stranded template molecules (Zdanowicz, 2010). . Following strand separation, the temperature of the thermocycler was reduced to 56ºC to allow the primers present in the reaction mixture to bind to the single-stranded DNA templates. This specific step is called Annealing, where one primer is designed to bind complementary to one strand of the DNA molecule in the forward direction (5 ' to 3 '), while the second primer is designed to bind complementary to the other strand of the DNA molecule in the reverse direction (3 ' to 5 ') (Zdanowicz, 2010). Once the two primers are annealed to the single-stranded DNA molecules, the process of Extension can occur. In this step of the PCR reaction the thermocycler was set up to 72 ºC. During Extension DNA polymerase extends the primers by adding dNTPs (deoxynuleoside trisphosphates),