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    Sbia022 Bioenergetics and Intermediary Metabolism

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    2023422047
    This document describes an experiment to determine the melting temperature of DNA using UV absorbance measurements at varying temperatures. Calculations are shown to determine the concentration of DNA solutions. Absorbance readings were taken and a denaturing curve graph was plotted from which the melting temperature was calculated to be 100°C. The GC content was determined to be 74.88% from the melting temperature.

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    Sbia022 Bioenergetics and Intermediary Metabolism

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    2023422047
    9 views4 pages
    This document describes an experiment to determine the melting temperature of DNA using UV absorbance measurements at varying temperatures. Calculations are shown to determine the concentration of DNA solutions. Absorbance readings were taken and a denaturing curve graph was plotted from which the melting temperature was calculated to be 100°C. The GC content was determined to be 74.88% from the melting temperature.

    Original Description:

    LTRAVIOELT ABSORBANCE OF DNAHYPERCHROME EFFECT

    Original Title

    SBIA022 BIOENERGETICS AND INTERMEDIARY METABOLISM

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    This document describes an experiment to determine the melting temperature of DNA using UV absorbance measurements at varying temperatures. Calculations are shown to determine the concentration of DNA solutions. Absorbance readings were taken and a denaturing curve graph was plotted from which the melting temperature was calculated to be 100°C. The GC content was determined to be 74.88% from the melting temperature.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    9 views4 pages

    Sbia022 Bioenergetics and Intermediary Metabolism

    Uploaded by

    2023422047
    This document describes an experiment to determine the melting temperature of DNA using UV absorbance measurements at varying temperatures. Calculations are shown to determine the concentration of DNA solutions. Absorbance readings were taken and a denaturing curve graph was plotted from which the melting temperature was calculated to be 100°C. The GC content was determined to be 74.88% from the melting temperature.

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    DEPARTMENT OF BIOCHEMISTRY, MICROBIOLOGY AND BIOTECHNOLOGY

    MODULE : BIOENERGETICS AND INTERMEDIARY METABOLISM

    MODULE CODE : SBIA 022

    GROUP NUMBER : 09

    EXPERIMENT 01

    TITLE : ULTRAVIOELT ABSORBANCE OF DNA-


    HYPERCHROME EFFECT

    SURNAMES & INITIALS : MAYIKO RR

    STUDENT NUMBER : 201727601

    DATE : 10 September 2021


    Calculations of how much is needed to dissolve DNA

    1.

    x 0,05mg
    =
    20ml 1ml
    × = 1mg
    3g 1mol
    = 1mg X 100mg X 650g

    = 4,62 X 10−6 mol


    1mg is needed to dissolve DNA to prepare 20ml
    of 0,05mg/ml

    2. 𝑥 0,015𝑚𝑔
    =
    30𝑚𝑙 1𝑚𝑙

    X= 0,45 mg
    1𝑔 1𝑚𝑜𝑙
    = 0,45mg X 1000𝑚𝑔 X 650𝑔

    = 6,92 x 10−7 mol

    3. To prepare 0,015mg/ml of DNA solution add 1,5 ml of NaCl to a volumetric flask


    containing 2,0 ml of DNA solution of a buffer
    4. THERMAL DENATURATION RESULTS TABLES AND GRAPHS

    4.1 TABLE 1: THERMAL DENATURATION RESULTS

    Tube number 1 2 3 4 5 6 7 8 9 10
    Temperature (oC) 25 50 60 70 80 85 90 95 100 100
    Abs 1 (260nm) 0.172 0.114 0.201 0.377 1.259 1.505 1.623 1.602 1.503 0.186
    Abs 2 (260nm) ,0,174 0.112 0.302 0.391 1.257 1.511 1.612 1.601 1.498 0.186
    Abs ave. (260nm) 0.172 0.113 0.252 0.384 1.258 1.508 1.618 1.602 1.501 0.186

    FIGURE 1: A graph showing Denaturing curve of DNA

    DENATURING CURVE OF DNA


    1.800
    1.600
    1.400
    ABSORBANCE (nm)

    1.200
    1.000
    0.800
    0.600
    0.400
    0.200
    0.000
    0 20 40 60 80 100 120
    TEMPERATURE (Oc)

    Abs ave. (260nm)

    5. y = 0.0248x - 0.8715
    6.
    Tm=69.3+0.41(G+C)
    Tm=100
    100=69.3+0.41(G+C)
    100-69.3=0.41(G+C)
    30.7/0.41=0.41 (G+C)/0.41
    G+C=74.88%

    7. (G+C)+(A+T)=100%
    (A+T)=100%-(G+C)
    (A+T)=100%-74.88%
    (A+T)=25.12%

    8. In this experiment, Tube 10 was used as a control to allow the us to be able to


    distinguish the effect of temperature in the experiment, to minimize the effects of
    other factors than the one being tested, which is temperature.

    9. Thermal denaturation is the process where a double helix or stranded DNA is broken
    down into single strands, the breaking of the double strands need temperature
    where the solution containing DNA is heated and the double stranded DNA unwinds
    and the hydrogen bonds between the nucleotides becomes weak and break
    because of the heat, the hydrogen bonds in the DNA strand plays a huge role in
    combining the nucleotides bases together, it binds the nucleotides bases together.
    The temperature at which half of the DNA molecules are denatured is called the
    Melting Temperature (Tm) of DNA. During the experiment a borate buffer was added
    into all the test tubes including the blank test tube, the borate buffer was added just
    to maintain the PH of the solution in all the test tubes.

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