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DNA Extraction - DOC

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					Hana Hakami                                                              2011


                             DNA Quantification
Introduction

      When working with DNA, it is important to know its
concentration. The easiest means of determining DNA concentration is
through spectrophotometric analysis. The concentration of DNA will be
expressed in units of mass per volume; for example: ng/ml or µg/ml.

First: Detremination of DNA Concentration

      There are more types of spectrophotometer and cuvette, but we
will use GeneQuamt spectrophotometer with quartz cuvette, and
NanoDrop Spectrophotometer.

Principle

       Nucleic acids, like many other substances, have the property of
absorbing light at a specific wavelength. Since nitrogenous bases absorb
UV light, the more concentrated the DNA solution, the more UV light it
will absorb.

      DNA and RNA absorb light maximally at a wavelength of 260 nm.
Because of this property, they can be quantified spectrophotometrically
with UV light source.

      The concentration of pure double-stranded DNA with an A260 of
1.0 is 50 µg/ml. In order to there is a linear relationship between
absorbance and DNA concentration, we can use Thus, one can use the
following formula to determine the DNA concentration of a solution:
            Unknown µg/ml = 50 µg/ml x Measured A260 x dilution factor

       The purity of a nucleic acid solution can be determined by
calculating the A260/A280 ratio. The nucleic acid absorbs maximally at
260 nm and protein (a principle contaminant) absorbs maximally at 280
nm.
                     Pure DNA has an A260/A280 ratio of 1.8.

                      Pure RNA has an A260/A280 ratio of 2.0.
Hana Hakami                                                         2011


      Cuvettes may be made of plastic, glass, crystal or quartz. The
choice of cuvette depends on the wavelength at which the absorbance is
to be measured. Glass or plastic cuvettes can be used at wavelengths
greater than 340 nm whereas quartz cuvettes are used in the ultraviolet
range because plastic and glass absorb UV light.

1. Determination       of   DNA     concentration    by     GeneQuamt
spectrophotometer

Materials

Spectrophotometer - Quartz Cuvette - Disttelted Water - TE Buffer - DNA
Templates - Microfuge tubes - Micro pipettes - Tips - Gloves – Biohazard
Bags & Container

Protocol

   1. Prepare your blank in microfuge tube as following:
          98 µl d.d.H2O + 2 µl DNA Hydration Solution (TE Buffer).
   2. Prepare your DNA samples in microfuge tubes as following:
                     98 µl d.d.H2O + 2 µl DNA Template.
   3. Mix them well.
   4. Turn on the Spectrophotometer (chose 50 as dilution factor-
      why?).
   5. Transfer the blank to Cuvette and read blank.
   6. Now, after blank wash the cuvette by d.d.H2O
   7. Transfer the first sample to cuvette and read sample.
   8. Record the reading of DNA concentration and the ratio of
      A260/A280.
   9. Wash the cuvette with d.d.H2O .
   10.Repeat steps number 7, 8 and 9 for every sample.

2. Determination       of    DNA     concentration     by    NanoDrop
Spectrophotometer

Materials

NanoDrop Spectrophotometer - d.d.H2O - TE Buffer - DNA Templates –
Kimwipes Tissue - Micro pipettes - Gloves – Biohazard Bags & Container
Hana Hakami                                                      2011


Protocol

   1. Turn on the NanoDrop Spectrophotometer and its software.
   2. Open NanoDrop program and press on nuclic acids button.
   3. Clean the surface of NanoDrop spectrophotometer by use d.d.H2O
   4. Add your blank as following:
                  1 µl DNA Hydration Solution (TE Buffer).
   5. Read the blank by press on Blank button.
   6. Clean the surface of NanoDrop spectrophotometer by use d.d.H2O
   7. Add the first DNA sample as following:
                            1 µl DNA Template.
   8. Read the concentration of DNA by press on Measure button.
   9. Clean the surface of NanoDrop spectrophotometer by use d.d.H2O
   10.Repeat steps number 7, 8 and 9 for every sample.
   11.Take the readings of DNA concentrations.



Second: Preparation of Working DNA

       When working with DNA in any biotechnology as PCR, you must to
use same concentration for all DNA samples. The DNA on this
concentration is called “Working DNA”. Choice of working DNA
concentration depends on many factors like, the concentrations of DNA
stock, type of biotechnique and experience of searcher.

     We will choose 50 µg/ml or 25 µg/ml as working DNA
concentration by use the following formula:
                            C1 x V1 = C2 x V2

				
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