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LAB 4

Quantitative Estimation of DNA /RNA


sample using spectrophotometer
Spectrophotometer:
It’s an instrument that measures the amount
of light absorbed by a solute in a solution, so
the more concentrated the solute, the more
light should be absorbed.

Spectrophotometer
Blank: is the solution that contains every
thing in your sample except the substance
that you are trying to measure or analyze.

Sample: is the solution that contains the


substance for which the concentration need to
be determined.
Principle
Concn. of nucleic acid Optical Density (OD)
(DNA or RNA) ∝ at λ 260nm

Optical density of DNA / RNA:

The intense absorption is due primarily


to the presence of aromatic rings in the
purine and pyrimidine bases
• Dilution factor =1/dilution
• Dilution =volume that you take from the
sample /final volume

• For example if you take 5 μ l from your


dna sample then add 495 water
• Calculate the dilution ,dilution factor
SUMMARY
5μl :495 μl
Horizontal gel electrophoresis
Gel electrophoresis
Gel electrophoresis:
is a powerful tool for the separation of
macromolecules with different size and
charges.
Electrophoresis
is the migration of charged molecules in
an electric field
Horizontal gel electrophoresis

? ?
Horizontal gel electrophoresis
Horizontal gel electrophoresis
Practical session of Agarose
gel electrophoresis
1-Preparation of agarose gel

TAE
Agarose buffer
Preparation of 1% agarose gel
in 50 ml

• 1 g 1oo ml Buffer eg TAE should be


1x
• ?? 5o ml
X=0.5 g
Microwave
oven
2- Staining the Gel

CAUTION! Ethidium bromide is a


powerful mutagen and is moderately
toxic.

Gloves should be worn at all times


STOCK 10 mg/ml
FINAL 0.5 mg/ml.
Wait for the gel to cool
3- Gel casting
Electrophoresis equipment
Pouring the agarose solution into the casting tray

»Avoid air bubbles


»Each of the gel combs should be
submerged in the agarose solution.
• When cooled, the agarose polymerizes,
forming a flexible gel.
• Carefully remove the combs
• Add enough electrophoresis”Running
buffer” to cover the gel. Make sure each
well is filled with buffer.
4-Sample Preparation

• Mix the samples of DNA with the


• (6x loading buffer ).
• WHY should we use:
• Glycerol?
• Bromophenol blue?
• Ethidium Bromide ?
5-Loading the samples

• Carefully place the pipette tip over a well


and gently expel the sample. The sample
should sink into the well. Be careful not to
puncture the gel with the pipette tip
6-Run the gel electrophoresis

• Place the cover on the electrophoresis chamber,


connecting the electrical leads to the power
supply.
• Turn on the power supply
• Higher voltage cause molecules to move faster
After the current is applied, make sure the Gel
is running in the correct direction.
Bromophenol blue will run in the same
direction as the DNA
DNA Ladder Standard

• Inclusion of a DNA ladder (DNAs of know sizes)


on the gel makes it easy to determine the sizes
of unknown DNAs.
7-analysis

UV transillimunitator

Gel documentation
• What is the difference between
transilluminator and gel documentation
system?!!
COMMENT

Smear “ degraded Intact DNA


DNA”
Quantitative estimation of RNA using Denaturated Agarose
Gel Electrophoresis

is used to check the size and integrity of


RNA
Quantitative estimation of RNA using Denaturated Agarose
Gel Electrophoresis
Visualization of RNA on Denaturing Agarose Gel
Electrophoresis

• Intact total RNA run on a denaturing gel will have


sharp 28S and 18S rRNA bands (eukaryotic samples)
• The 28S rRNA band should be approximately twice
as intense as the 18S rRNA band. This 2:1 ratio
(28S:18S) is a good indication that the RNA is intact.
Estimation of RNA using Native Agarose Gel
Electrophoresis

• Native agarose gel electrophoresis may be


sufficient to judge the integrity and overall quality of
a total RNA preparation by inspection of the 28S and
18S rRNA bands.

• The secondary structure of RNA alters its migration


pattern in native gels so that it will not migrate
according to its true size
• Bands are generally not as sharp as in denaturated
gels, and a single RNA species may migrate as
multiple bands representing different structures.

•Run the gel at 5-6 V/cm measured between the


electrodes.