DNA


Deoxyribonucleic acid (DNA) is a molecule
that encodes the genetic instructions used in the
development and functioning of all known
living organisms and many viruses.

Along with RNA and proteins, DNA is one of the
three major macromolecules essential for all
known forms of life.

Isolation of DNA from human
blood

Human blood contains several types of
cells such as RBC’s and WBC’s these
are present in blood in different amount
according to their requirement and in
this we can isolate DNA from the WBC’s
as they contains nucleus while prior one
does not.

REQUIREMENTS:

a.
b.
c.
d.
EXTRACTION BUFFER
10 mM Triscl (pH 8.0)
0.1 mM EDTA (pH 8.0)
20 µgm/ml Pancreatic RNase
0.5% SDS
PBS
Nacl - 0.8 gm
Kcl – 0.2 gm
KH2PO4 – 0.24 gm
Na2HPO4 – 1.4 gm
Dissolve in 800 ml DW maintain the pH at 7.4 with Hcl and
autoclave the solution.
 chloroform : isoamyl alcohol (24:1) make 10 ml in volume.
 Saturated phenol : chloroform : isoamyl alcohol (25:24:1) Take
5 ml of mixture no.3 and add 5 ml of saturated phenol.

a.
b.
c.
d.


Procedure

Sample is taken from the patient and stored in
a vial coated with the EDTA(anti-coagulant)

From the sample 500 µl blood is pipette out
and mixed with the 500 µl PBS.

The sample is then centrifuged at 5000 g for
10 min at 4°C.

The supernatant is taken out and pellet is
mixed with 200 µl extraction buffer and 1 µl
RNase, This is then incubated at 37°C for 1 hr.


add 200 µl of EB and 600 µl of saturated phenol in Aq.
Layer and centrifuge it at 12000 rpm for 10 min.

Step no. 6 was repeated twice without the EB and then
supernatant is taken in fresh microcentrifuge tube.

600 µl of chloroform : isoamyl alcohol (24:1) is added in
the supernatant and it is centrifuged at 12000 rpm for 10
min, this step is repeated thrice.

Supernatant is taken in fresh microcentrifuge tube and
mixed with 600 µl of Saturated phenol : chloroform :
isoamyl alcohol (25:24:1) then it is centrifuged at 12000
rpm for 10 min, this step is repeated twice.

Now the supernatant is mixed with 60 µl of ammonium
acetate and left overnight at -20°C for precipitation.


After precipitation it is centrifuged at 12000 rpm for 10 min
at 4 °C.

1 µl of proteinase-k is added and then it is incubated at
50°C for 3 hr.

Now for washing supernatant is discarded and the pellet
is mixed with 70 % ethanol and centrifuge at 12000 rpm
for 10 min and then tubes were decanted and kept on RT
for drying for 4 hr.

After tubes are completely dried the pellet is dissolved in
the 30 µl of TE buffer and incubated for 2 hr at 37°C.

Visualisation

For visualising the DNA we prepared 25 ml of
0.8% agar gel and casted it in the electrophoretic
unit.

To load in the wells 10 µl of sample is mixed with
the 3 µl gel loading dye and run at 75 mV for 1.5
hrs.

The
bands
are
transilluminator.
visualized
using
UV

DNA


It is done to check the mutation present in the DNA
sample, and to identify the mutation sites we have to use
a particular restriction enzyme as in BRaf gene which is
present in MAP kinase pathway V600E mutation is very
common in papillary thyroid carcinoma.

If the mutation has taken place then the site for the RE
will be depleted in the DNA and it will give single band but
if it is having no mutation then this will result in the three
bands as site for RE will be present.

REQUIREMENTS
2 µl of buffer (tango)
 10 µl of DNA sample
 1 µl of restriction enzyme
 7 µl of RNase free water
Mix these to make the total volume 20 µl and take in
microcentrifuge tubes
 25 ml of 3% agrose gel with 4 µl of ETBr in 1X
TBE.
 Molecular ladder: gene ruler 50 bp (1 µg/ µl).


PROCEDURE

The prepared 20 µl solution
incubated for overnight.

Then cast the gel in the electrophoretic unit and pre run
it for 5 min for at 20 mV.

Load the 8 µl of incubated solution with 2 µl of gel
loading dye.


Also load the 5 µl of ladder then run it at 70 mV for 2.5
hrs.

Now the gel is observed in the UV transilluminator.

Samples 1,2,3,4
237 bp
117 bp
87 bp
33 bp
ladder

DNA


This technique can be used for many
applications but mainly it is used for the
amplification of DNA.

It uses polymerase enzyme and primers to
replicate the desired DNA sequence.

REQUIREMENTS
Microtiter plate
 PCR kit
 PCR machine


PROCEDURE

The sample is taken in the plate
provided with the machine.

A control sample is also used generally
some housekeeping gene like betaactin.

Now the primers are added and the
polymerase enzyme too.


Load the plate in the machine and set the
plate profile in the software.

Now after the process is finished retrieve the
plate with amplified sample.

VISUALISATION

3% agrose gel.

PCR products are loaded in the wells
with the gel loading dye.

A ladder is also used for conformation of
the size of product.

Products of PCR
Primer
1,2,3,4,5,6,7
Ladder

RNA

RT-PCR
THIS IS A TECHNIQUE THROUGH WHICH WE CAN
AMPLIFY THE SELECTED cDNA STRANDS WITHIN A
VERY SHORT TIME SPAN.
IN THIS WE CAN VISUALIZE THE AMPLIFICATION IN
REAL TIME WITH THE HELP OF MACHINE.

REQUIREMENTS
 cDNA
 rt-PCR kit
 PCR well plate
 Trizol (phenol + guanidine thiocyanate)
 Tissue sample
 Homogenizer
 Sybr green master mix

RNA EXTRACTION( TRIZOL METHOD)
 Homogenization:
tissue preserved in liq. N2 is homogenized in Trizol reagent.
 Phase separation:
Incubate for 5 min then add 200 µl chloroform/1ml Trizol
incubate for 3 min then centrifuge at 12000g for 15 min at
4 C.
 Upper aq. Phase contains RNA.
 1-bromo-3-chloropropane.

 RNA precipitation:
Mix isopropyl alcohol 0.5 ml/1ml Trizol
Centrifuge at 12000g for 10 min at 4°C
 RNA wash:
mix pellet with 1ml ethanol (70%)/1ml Trizol
Centrifuge at 7500g for 5 min at 4 C

 Resolving the RNA:
Dry the pellet
Dissolve in 50µl RNase free water
Store at -80 °C

Convert the RNA into cDNA
Take 500 ng to 2µg of the converted sample
Add in rtPCR plate along with Sybr green master mix and distill
water
Start the program in machine and then see the amplification in real
time
Obtain the amplified sample

TPI


It is the building block of our body. It is present in
tremendous variety and play several roles altogether in
our body. And to study any change in any organism’s
body we can go for checking the protein’s nature and
amount.
To check the level of expression in the body of any
organism we can opt for following techniques:

Western blotting.

Immunohisto chemistry.

PROTEIN

 This
technique is used for the separation of
proteins on the basis of their molecular weight.
 level
of expression with the help of any reaction
like Ag-Ab interaction and developing the blot on
x ray film with the help of any chromogenic
substrate.

 Gel
casting unit
 Chemicals for the PAG, separating gel will be of
6-15%
 Running buffer (pH 8.3)
 Transferring buffer (pH 8.5)
 Nitrocellulose membrane
 Transfer unit
 Current supply unit

 Prepare
the unit and buffers.
 Now
put the unit in the electrophoretic tank
and add running buffer.
 Load
the equal amount of the protein samples
(in micrograms) with loading dye.
 Attach
the current supply unit and let it run
until dye has reached the bottom.


On completion remove the gel and assemble the transfer
unit and transfer the proteins on nitrocellulose membrane
with help of transfer buffer.

After transfer has completed take the membrane and use
ponctuS dye to stain the proteins, it is not permanent
hence after visualisation it can be washed.

Prepare 5% blocking solution (milk) in TBST/PBST, and
give exposure for 1 hr.

We prepared the primary antibody in 1-5%BSA diluent
and add to the membrane. This process will take place
over night in the cold room at 4°C.

 Next
day the membrane is again washed with
the TBST three times for at RT for 15 min each.
 The
membrane is washed using TBS solution on
Rocking platform. Three washes of 15 min each
will be given.
 Then
secondary antibody is diluted in the
blocking solution and added to the membrane
and the exposure given will be of 1 hr.
 After
that again washing with the TBST three
times and then blot development procedure.

 In
Blot development we take the membrane out
and add the substrate which is having luxogenic
reaction.

The time of reaction taking place is moderated
according to the individual and after the
exposure of X-ray film, it will be put in the
machine for development of blot.
 The
intensity of the blot is proportional to the
level of expression.
 Luminol+H2O2
is the substrate.

PROTEIN

 This
technique is used for checking the
localisation of any particular protein at a
particular time in the cell.
can’t quantify the protein hence generally done
parallel with the western blotting. It is very
important as it can show the effect of any
disease or any compound on a particular protein.
 It
















Prepared blocks of desired tissue
Water bath
Poly lysine coated slides.
Xylene
Alcohol (100%, 90%, 70%, 50%)
PBS
Citrate buffer
Na2BH4
H2O2
Blocking solution (5% sheep serum, 0.1% triton)
DPX
Primary antibody
HRP labelled Secondary antibody
Streptavidin-biotin complex
Pressure cooker


Place the block on the mount and align it to the
blade.

Adjust the section size and remove the extra wax.

Switch ON the water bath, and start cutting the
sections.

Now the section will be obtained and we can fix
them on the polylysin coated slides.

Deparaffinised on hot plate at 60°C.

 The
slides are placed in the coupling jar with
Xylene and jars are put into the hot air oven at
60°C. After 10 minutes use new Xylene jar.
 Then
rehydration is done by keeping the slides
into the alcohol and slowly decreasing the
concentration of the alcohol at last it will be
washed into the distil water for 5 min.
 Then
min.
the slides will be transferred into PBS for 5


Then transfer the slides into the citrate buffer and transfer the jar into a
pressure cooker with water at 120°C this will be kept for about 45 min.

Now the slides were cooled down and then transferred to the jar with
PBS for three wash10 min each.

1% Sodium borohydrate (Na2BH4) is prepared and the exposure to the
slides is given in the dark.

Again wash the slides with PBS two times 10 min each.

Then we prepare the blocking solution add it to the slides and keep the
slides covered with parafilm.

After this we will give the exposure of primary antibody (1:500) and keep
them covered for overnight in medicool at 4°C.


Next day after bringing the slides at RT we wash them
with the PBS three times 5 min each.

Then slides are transferred in 3%H2O2 freshly prepared
for 20 min.

Then again wash with PBS three times 10 min each.

Take out the slides on the slide chamber and expose
them to the secondary antibody with sheep serum and
1X PBS for 60 min.

Again the slides are washed with PBS three times 10
min each.


Now take out the slides and add the substrate for 15 min.

Wash with PBS three times each of 5 min.

Now the exposure of DAB is given to intensify the signal for 20
min.

Wash with PBS and after drying mount with DPX this
increases the refractive index.

Now seal them with transparent paint and leave them for
drying.

When completely dried observe under the Phase Contrast
Microscope. This will show the specified protein.

Antigen Aquaporin II in kidney cell through phase contrast
microscope at 10X

Antigen Aquaporin II in kidney cell through phase contrast
microscope at 40X

PROTEIN

 It
is done to obtain the section of any tissue from
any organ of an organism.
 This
block is used in immunohistochemistry.

 Select
out the subject animal and sedate it with
the chloroform.
 Then
cut out its head and retrieve the brain with
the help of dissection kit and store it in the PFA.

After 24 hrs we go for dehydration of the tissue.

50% alcohol
1.5hrs
70% alcohol
1.5hrs
80% alcohol
1.5hrs
90% alcohol
1.5hrs
100% alcohol
1.5hrs
100% alcohol
1.5hrs
100% alcohol
1.5hrs or over night

After last step we leave it for the night and then use chloroform to replace
the alcohol
chloroform
1.5hrs
chloroform
1.5hrs
chloroform
1.5hrs

We use molten wax to totally occupy the free spaces
within the tissue so that it retains its structure.
Wax
1.5hrs
Wax
1.5hrs
Wax
1.5hrs

 After
the final exposure transfer the tissue at the
base of mould and pour the molten wax on it
then set on the cold plate and cover the mould
with the casket.
 After
4°C.
it solidifies remove the mould and store at

BRAIN TISSUE FROM MICE

PROTEIN ESTIMATION (BRADFORD METHOD)

 Estimation
of protein content in any sample
provided either by patient or obtained through
animal model tissue is important as according to
the concentration present we will be able to
identify the protein’s molecular weight and use it
in western blotting.

 Protein
standard sample
 PBS
 Bradford
dye
 Spectrophotometer
 Protein samples

 First
we prepared a working stock solution of 100
µg/ml from 1.34 µg/ml.
 From
this working stock we prepared standards
of different concentration:
 0.8
µg/ml
 1.6 µg/ml
 4.0 µg/ml
 6.0 µg/ml
 12.0 µg/ml
20 µl WS + 1980 PBS
40 µl WS + 1960 PBS
100 µl WS + 1900 PBS
150 µl WS + 1850 PBS
300 µl WS + 1700 PBS

 Now
we mixed 200 µl dye with 800 µl of all these
standards and also prepared blank by taking 800
µl PBS in place of standards.
 The
samples which are to be examined are taken
800 µl and mixed with 200 µl of dye.
 Now
we started taking readings from UV
spectrophotometer.
 Thus
obtained readings will give a plot of conc vs
absorbance. Thus we can calculate the
concentration of unknown sample.

0.6
0.5
0.4
abs 0.3
abs
0.2
0.1
0
0
2
4
6
concentration
8
10
12

PRIMARY CULTURE

 This technique is important to study the behaviour
of cells towards a specific condition or compound.
 The tissue used in this is either obtained through
model organism or biopsy.

 Model organism
 Dissection kit
 PBS
 Media (DMEM) with phenol red
 Serum (calf serum)
 Filter assembly
 Sterilised glass wares
 6 well plate

 Put the serum for freeze thaw and after that filter
sterilize it.
 Dissolve the media in milliQ water and then filter
sterilize it.
 Model organism is dissected and its desired organ is
retrieved (in this case kidney).
 The organ is washed properly in PBS, bring it in
culture room.

 Neatly with the help of surgical blade cleave the kidney
transversely.
 Retrieve the cortex part of the kidney and discard the
remaining part.
 Take the tissues in PBS and prepare homogenate and
through repetitive pipetting make single cells.
 Equal volume of cells is added in the wells.

 Now the media is added in the cells and then serum is
also added.
 The cells are daily washed with PBS and media is
changed.
 To
observe the growth of cells inverted light
microscope is used.
 After it has taken up more than 80% volume of the
wells we can passage the cells.

visualisation
Kidney cells through
inverted miroscope

 This technique is important to study the cell on sub
cellular level.
 We can visualise these and
obtain information
regarding the cells.
 We check the level of any specific protein at any
specific location.

 Model organism
 Mitochondrial buffer
 centrifuge
Mitochondrial buffer
 1mM EDTA(pH 8.0)
 0.32 M sucrose
 10 mM Tris-cl(pH 7.4)
 1mM PMSF
 1mM DTT

 Dissect the model organism and retrieve the desired
tissue in this case cerebral cortex and Preserve it in the
liquid nitrogen.
 Protease inhibitor cocktail 5 µl/ml buffer, weigh 100
mg tissue and take it in 1ml mitochondrial buffer.
 Homogenize in power homogenizer then keep on ice
for 15 min.

 Centrifuge at 700 rpm for 10 min and take out the
supernatant.
 The nuclei and unbroken cells were pellet down by
centrifuging at 2000 rpm for 10 min.
 Thus obtained pellet is nuclear fraction (A) add buffer
and store at -20 °C.
 The obtained supernatant is centrifuged at 2000 rpm
for 10 min.
 Thus obtained pellet is nuclear fraction (B) add buffer
and store at -20 °C.

 The obtained supernatant is centrifuged at 2000 rpm
for 15 min.
 Thus obtained pellet is mitochondrial fraction (C) add
buffer and store at -20 °C.
 The obtained supernatant is centrifuged at 2000 rpm
for 15 min.
 Thus obtained pellet is mitochondrial fraction (D) add
buffer and store at -20 °C.
 The remaining supernatant is designated as cytosolic
fraction (E), add buffer and store at -20 °C.


This procedure is very important as it prepares the
model organisms on which experiment models can
be executed. These are kept in a very specific
condition and given specific diet so that their
growth and development pattern can be
controlled.

On the basis of desired condition of these
organisms we prepare a model according to
which they are gradually brought to that condition
and then their condition is analysed by various
tests.


These are having a life span of about 1 year
but their various growth stages are required
hence breeding is required and it is also
monitored.

For various experimental models like induced
nephropathy(diabetic), hyperthyroidism and
hypothyroidism different set of diet and
growth stages are required hence they are
grown separately.


Condition of parents is monitored constantly
and their diet is also regulated according to
the model.

Every morning we have to check the urinary
sample for sign of fertilisation if sperm is found
then these are separated and time is noted
down.

Thus different embryonic stages can be
obtained and we can check for the level of
effect of any particular exposure that was
given to the parents.


To preserve adult organism’s organs and serum we
have to set a different model involving different
kind of parameters.

Their diet is kept regulated and also we have to set
a control organism to compare the growth.

Daily changing of bedding and sanitation is
important as we don’t want to affect the
environmental condition.

Giving daily set dosage of medicine is also very
crucial.


After completing growth model we have to
procure the organs and blood from that
organism and after that we preserve organs
either in liquid nitrogen(for western blotting) or in
PFA(block preparation and IHC).

For taking out the blood properly we have to
keep the organism alive but sedated so that his
heart keeps pumping and we would be able to
extract the blood and from that we can isolate
serum.


If we have to preserve bones
(vertebrae, femur) to check the effect of
Osteocalcin and RNA analysis we also
preserve them in PFA.

Different embryonic stages are marked as
E16, E18 according to their growth stage.

These are nocturnal hence are kept in dark
and quite room.

If we bring them out from dark room their
hormonal level will change so before going
for dissection we have to let them stabilize.

Embryo of 18 days


If we are inducing any medical condition in the
organism then we have to closely monitor the
changes in the health of the subject. Like in
case of diabetic condition we have to check
for blood glucose level.

To check their level of muscular activity we use
ferries wheel and thus we can find out which
subject is doing good and whose condition is
going bad.

Proper discarding of the dissected subject is
also important.

Always use sterile kit for dissection and keep all
the necessities ready.

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