Quantitative estimation of DNA and RNA

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Quantitative estimation of DNA and RNA
Estimation of nucleotides is the very important
step after sample isolation to find out the
amount of the nucleotide present and to check
for the suitability of the sample for the further
analysis.

• ?

• Related Loss DNA properties
• gt Prior Viewing IDD-1. Extraction of
  bacterial protein, IDD-6. Extraction of serum
  protein
• gt Future Viewing IDD-17. SDS-PAGE, IDD-33.
  Western blot assay
• Course Name Quantitative estimation of DNA and
  RNA test
• Level(UG/PG) UG
• Author(s) Dinesh Raghu, Vinayak Pachapur
• Mentor Dr. Sanjeeva Srivastava

The contents in this ppt are licensed under
Creative Commons Attribution-NonCommercial-ShareAl
ike 2.5 India license
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Learning objectives
1

• After interacting with this learning object, the
  learner will be able to
• Describe the presence of DNA/RNA in the sample
• Define the mechanism of detection
• Operate the steps used in colorimetry
• Infer the law governing the colorimetric analysis
• Assess the troubleshooting steps involved in the
  experiments.

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Definitions and Keywords
1

• DNA Deoxyribonucleic acid, contains the genetic
  information used in the functioning and
  development living organisms
• 2. RNA are ribonucleic acid, chemical nature is
  very much similar to DNA, play an active role in
  cell signaling, carry out biological reactions
  and even control expression gene.
• 3. Ethidium bromide a fluorescent tag binds
  reversible to DNA molecule which fluoresce with
  orange color when exposed to UV.

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Master Layout
1
Slide 5-10
Gel Casting
Slide 11-12
Making the standards and sample
2
Slide 13-15
Agarose gel electrophoresis
Slide 16-18
3
UV transilluminator
Slide 19-20
Making the standards and sample
4
Slide 21-26
Spectrophotometer
Slide 26-29
Calculate the concentration
5
5
Step 1
1
T1Gel casting
2
Beaker
Magnetic bead
3
Description of the action
Audio Narration (if any)?
Show magnetic stirrer instrument. Let user place
the beaker on it. Display the beaker containing
powder at bottom, liquid layer on top and a
magnetic bead at the bottom. Instruct user to ON
the instrument, let user cotrol the speed nob and
regulate it accordingly to control the mixing
speed in the beaker. Animate powder getting into
the solution. Show a turbid solution turning
colorless
4
The magnetic stirrer helps for a even mixing of
solute Into the solvent at faster rate.
5
6
Step 1
1
T1Gel casting
2
Measuring balance
3
Audio Narration (if any)?
Description of the action
Show a measuring balance, with display, ON, OFF
and TARE/0 buttons on it. let user ON it, display
reading as 0.000g, let user picks up the paper
from the rack, makes 1/10 of folding on the sides
and places it on the balance. Now the display
reading changes to 0.003g. Instruct user to TARE
the reading. And animate to click the tare
button. Once user clicks it, reading must show
0
When measuing with paper, the weight of the paper
need to be tared from actual reading.
4
5
7
Step 2
1
T1 Gel casting
EDTA stock
EDTA
2
Water
3
Instruct user to prepare stock solution of EDTA
buffer, let user take out EDTA bottle from the
rack and keep next to the balance. Let user weigh
93.05g of EDTA, transfer to a fresh beaker,
measure and add 400ml of water to the beaker, let
user makes a movement on the baker to dissolve
the reagent into the solution. With the help of
pH meter set the pH to 8.0 by adding NaOH and
later make the volume to 500ml by water.
Ethylenediamine tetra acetic acid (EDTA) stock
need to be prepared in advance.
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5
8
Step 3
1
T1 Gel casting
TAE stock
TRIS
2
Water
3
Instruct user to prepare stock solution of TBE
(50X) buffer, let user take out tris base and
glacial acetic acid bottle from the rack and keep
next to the balance. Let user weigh 242g of Tris
base, transfer to a fresh beaker, measure and add
750ml water to the beaker, let user makes a
movement on the baker to dissolve the reagent
into the solution and mixing done as in slide 5.
Instruct user to measure and add 57.1ml of
glacial acetic acid and 100ml of EDTA stock
solution. Let user make up the volume to 1000ml
by water.
The stock solution of TBE(50X) can be stored at
room temperature and pH adjustment is not
required. For running the gel prepare 1X TBE
buffer, which can be used for DNA quantification.
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5
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Step 4
1
T1 Gel casting
TBE stock
Tris
2
Boric acid
3
Instruct user to prepare stock solution of TBE
(5X) buffer, let user take out tris base and
boric acid bottle from the rack and keep next to
the balance. Let user weigh 54g of Tris base,
27.5g boric acid, transfer to a fresh beaker,
measure and add 900ml water to the beaker, let
user makes a movement on the baker to dissolve
the reagent into the solution. Instruct user to
measure and add 20ml of EDTA stock solution. Let
user make up the volume to 1000ml by water.
The stock solution of TBE(5X) can be stored at
room temperature and pH adjustment is not
required. For running the gel prepare 10X TBE
buffer, which can be used for RNA quantification.
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5
10
Step 5
1
T1 Gel casting
Buffer
2
Gel solution
Agarose
Description of the action
Audio Narration
3
Instruct user to take out agarose bottle from the
rack and keep next to the balance. Let user weigh
320mg of agarose and transfer to a fresh 250ml
conical flask. To the flask let user add 40ml of
TBE buffer. Instruct user to take the conical
flask for heating in oven for few seconds to
dissolve the agarose by opening the oven and
placing the flask and watching on the instrument.
To the melted agarose add 0.5ug/ml of ethidium
bromide. Label it as Gel solution.
Ethidium bromide is a fluorescent dye interacts
with base pairs of DNA/RNA and forms a complex.
The complex get absorbed and emitted which can be
acquired at visible spectrum.
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Step 6
1
T1 Gel casting
2
3
Instruct the user to put the gel solution into
the gel casting unit. Place the gel casting unit,
let user clean it with tissue, place the comb on
top of it, pour the gel solution slowly. Keep the
setup for 30min. Events must happen when the user
clicks on the hand. Show a change from liquid to
solid state
Place the gel casting unit, depending upon the
sample user can place the comb for the wells.
Pour the gel solution into the unit, avoid air
bubbles.
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Step 7
1
T2 Making the standards and sample
2
3
Once the gel is cast, let user place the gel in
the running unit, pour the 1X buffer to the
required level.
Add the running buffer to the unit with the
required concentration.
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Step 8
1
T2 Making the standards and sample
2
3
Instruct the user to prepare sample and standard
for the loading. Let user take out the dye,
sample and DNA/RNA standards from the freezer,
keep it on ice for 5min. In each well, let user
mix 1ul of loading dye(blue in color) with 2-3ul
of sample/standard. Animate the step, for user
taking a pipette setting it to 1ul, taking out
dye, pipette it on parafilm. Now similarly take
out sample/standard with pipette, mix it with the
dye solution on the paraffin with pipette, after
mixing load in the required well and let user
make a note of it.
The dye and the sample/standard need to be mixed
at proper ratio before loading into the gel. The
mixing step can be done on parafilm or on
eppendrof tube cap. Try to load standards in the
first well if possible followed by sample.
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Step 9
1
T3 Agarose gel electrophoresis
2
3
Let user connect the run unit to power supply,
let user make proper connection, check for the
buffer level, anode and cathode wiring. Make the
voltage set for 70V and click ON button.
Animate small bubbles coming from the electrode
and movement of the blue bands as time passes.
Set the required voltage, have a regular check
for the buffer level.
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Step11
1
T3 Agarose gel electrophoresis
2
3
Animate the movement of bands to the 1/3 of the
distance of the gel. Instruct user to stop the
unit, with help of gloves let user open the lid
of the running unit, pick up the gel and take it
for UV check.
After bands have been resolved in the gel, the
gel can be taken for UV visualization.
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Step12
1
T4 UV transilluminator
2
UV instrument
Gel on UV platform
gel view under UV
Scanned image
3
Show the UV instrument connected to monitor. Let
user place open the UV platform drawer, place the
gel on the UV platform, close it, let user On the
UV light. click on the UV software popup a
window, with File, view, edit, save options. Let
user click on viewgtpre-scan. Animate a small
window with image appearing row by row to show
the scanned image. Animate user to look at the
real image, with bands glowing like in gel view
under UV. Provide a option for save image, when
the scan is completed. Let user name the file
accordingly.
Scan the image at required parameters settings.
Let user makes a note of the bands and the
readings by saving the file.
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Step13
1
T4 UV transilluminator
2
3
Please re-draw the above figure. Figure a
represents the ethidium bromide. Animate in
figure b DNA molecule coming in contact with
ethidium bromide(small beads) getting stacked
between base pairs.
Ethidium bromide is a fluorescent dye interacts
with base pairs of DNA/RNA and forms a complex.
The complex get absorbed and emitted which can be
acquired at visible spectrum.
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Step14
1
T4 UV transilluminator
2
Scanned image
3
Animate the steps, user doing the analysis on the
scanned image by taking through the IDD for
SDS-PAGE gel analysis.
The analysis on the scanned image of the sample
bands can be quantified by comparing with that of
the standards. If the sample bands run along the
standard bands and are seen at the same position,
we can say that sample contains the DNA/RNA of
interest. If there are no bands within the marker
run length, we say no presence of DNA/RNA in the
sample.
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Step15
1
T1 Making the standards and sample
TAE stock
TRIS
2
Water
3
Instruct user to prepare stock solution of TAE
(50X) buffer, let user take out tris base and
glacial acetic acid bottle from the rack and keep
next to the balance. Let user weigh 242g of Tris
base, transfer to a fresh beaker, measure and add
750ml water to the beaker, let user makes a
movement on the baker to dissolve the reagent
into the solution. Instruct user to measure and
add 57.1ml of glacial acetic acid and 100ml of
EDTA stock solution. Let user make up the volume
to 1000ml by water.
The stock solution of TAE(50X) can be stored at
room temperature and pH adjustment is not
required. For running the gel prepare 1X TAE
buffer, which can be used for DNA quantification.
4
5
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Step16
1
T1 Making the standards and sample
2
3
Instruct user to take out the sample from the
-20C freezer, place them on ice for thawing for
5min. Let user set the pipeete to 1000ul to take
out TAE buffer and transfer to fresh tube, label
it as BLANK. Let user set the pipette to 10ul
to take out the sample in fresh tube, now let
user set the pipette to 900ul to add TAE buffer
to the tube and label it as SAMPLE. Let user
close the tube and vortex the tube for proper
mixing.
Sample for the quantification of DNA must be
taken out. After dilution with buffer give a
short vortex to mix the solution. Now the
solution is ready for the spectrometer reading.
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Step 17
)?
1
T2 UV-Visible spectrophotometer
Display
Options like number 0-9, set wavelength,
autozero, absorbance
2
Lid that can be opened
3
cuvette
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Step 17
1
T2 UV-Visible spectrophotometer
Animate the instrument as in figure and redraw
the instruments with the specification mentioned
in the figure and zoom the instrument and show a
schematic as shown in the figure with the
labelings but redraw completely
UV-Visible spectrophotometer has a monochromator,
light source and sample holder and detector,
Light from the source are converted to a
monochromatic light of particular wavelength and
allow it pass through the sample and amount of
light that emerges is detected by a detector.
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Step 17
1
T2 Spectrophotometer
2
3
Absorbance of a sample that is nothing but
logarithm ratio of incident light to reflected
light is equal to the product of path length,
epsilon constant and concentration of the
solution. Form the above equation one can
calculate the concentration of the solution given
the incident light, reflected light and path
length value.
Animate the above display, like Io passing
through the sample of concentration c,
travelling a path length l and coming out as
I. Now animate putting these parameters in the
equation above for user click to explain
accordingly.
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Step 18
1
T2 Spectrophotometer
Let the user take the tube 1 and take 2 cuvettes
as in figure . Instruct the user to press the
open lid show two opening inside it one after the
other in longitudinal way. Show like pouring the
contents from tube 1 to both the cuvettes , show
like taking the tissue and wiping on the sides
and placing it in the openings, now animate like
closing the lid and press absorbance . (before
keeping the cuvette the reading should be 0.000,
once the cuvette is kept and absorbance is
pressed it should be 0.123) Now instruct the user
to pressauto zero and the reading should be
0.000 and remove the cuvettes by opening the lid
and taking out the cuvette from opening 2 throw
the solution out from the cuvette
Auto zero and callibrate the instrument using the
control solution without hydrogen peroxide
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Step 22
1
T2 Spectrophotometer
Set the instrument at 280nm to set it at auto
zero with buffer and taking the reading for the
same sample at 260nm.
Repeat the same steps like in slide25 to set the
instrument at 280nm. This time animate the
reading of the sample around 2.3. let user makes
a note of the reading. Also user should take
reading at 260nm.
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Step 24
T3 Calculate the concentration
1

• A ratio value between 1.8-2.0 denotes, presence
  of nucleic acids.
• A ratio value lower than 1.8 denotes presence of
  proteins and/or other UV absorbers.
• A ratio value higher than 2.0 indicates that the
  samples may be contaminated with chloroform or
  phenol.
• In either case (lt1.8 or gt2.0) it is advisable to
  re-precipitate the DNA.

Instruct user to do the ratio calculation of
260/280. animate the ration being displayed on
the screen with audio narration.
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Step 25

• DNA concentration (?g/ml) OD260 x 100
  (dilution factor) x 50 ?g/ml
• 1000

• DNA concentration (?g/ml) OD260 x 100
  (dilution factor) x 50 ?g/ml
• 1000

• DNA concentration (?g/ml) OD260 x 100
  (dilution factor) x 50 ?g/ml
• 1000

• DNA concentration (?g/ml) OD260 x 100
  (dilution factor) x 50 ?g/ml
• 1000

T3 Calculate the concentration
1
DNA concentration (ug/ml) OD at 260 x 100
(dilution factor) x 50 ug/ml

1000
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3
DNA concentration can be calculated by the above
formula.
Instruct user to do the DNA concentration
calculation. animate the calculation being
displayed on the screen with audio narration.
4
The calculated concentration can now taken for
further analysis, for more information look for
future IDD.
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Slide 13-15
Slide 16-18
Slide 5-10
Slide 11-12
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage

• Animation area
• In slide-17 provide user a gel image with bands
  in sample well appearing after the DNA standards
  bands. Let user interrupt the result?
• Instruction user must come out with the solution
  pointing out presence of proteins and no DNA/RNA
  in the sample. Must go back to sample extraction
  step to make the changes.

Interactivity area
Instructions/ Working area
Credits
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Slide 26-29
Slide 19-20
Slide 26-29
Slide 19-20
Slide 21-26
Slide 19-20
Slide 21-26
Slide 21-26
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage

• Animation area

Interactivity area
Instructions/ Working area
Credits
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Questionnaire
APPENDIX 1

• Question 1
• Native DNA, molecular weight
• Greater than (40 kb)
• less than (40 kb)
• Greater than (20 kb)
• Greater than (80 kb)
• Question 2
• the bound dye itself absorbs radiation at
• a) 366nm
• b) 460nm
• c) 480nm
• d) 302nm and 366nm
• Question 3

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Questionnaire
APPENDIX 1

• Question 4
• In either case 460/480 ratio value (lt1.8 or gt2.0)
• re-precipitate the sample.
• precipitate the sample.
• Mix the sample
• Add buffer
• Question 5
• As the absorbance increases , the intensity of
  the outgoing light
• Decreases
• Increases
• Remains same
• zero

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APPENDIX 2
Links for further reading

• Reference websites
• http//www.youtube.com/watch?v6mQGNDnOyH8feature
  related
• Book
• Hoisington, D. Khairallah, M. and
  Gonzalez-de-Leon, D. (1994). Laboratory
  Protocols CIMMYT Applied Biotechnology Center.
  Second Edition, Mexico, D.F. CIMMYT.

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APPENDIX 3
Summary
The method mostly involves the quantitative and
qualitative estimation of DNA/RNA using
absorbance ratio method and by agarose
electrophoresis. The quantification and
estimation of the DNA/RNA can be depend lot on
standards and gel concentration used.