Name REEMA AMIN Registration no FA19-BSO-058

Videos description
About pipetting we learned how to use
pipette correctly, it has got a control button,
ejector and two stop points, The operator
depresses a plunger that moves an internal
piston to one of two different positions. The
first stop is used to fill the micropipette tip,
and the second stop is used to dispense
the contents of the tip. we apply light
pressure to insert pipette into the tip when
escalating two factors influence accurate
liquid uptake the aspiration angle and
emersion depth the exploration angle
should be vertical otherwise too much liquid
is aspirated, emersion depth must be as small or as large according to the requirement. Pipettes
were developed for the medical and laboratory industries but now they are used in beverage
and cannabis industries.

Agarose Gel Electrophoresis

Second video was explaining agarose gel electrophoresis, it was about loading and running
DNA sample on agarose gel.it has a Mini sub cell connected with electrodes (the most important
and expensive part of whole apparatus) It passes current which separates DNA sample. DNA
sample is loaded at one end of a gel matrix (usually agarose or acrylamide) that provides a
uniform pore size through which the DNA molecules can move. Application of a constant electric
field causes DNA fragments ) to migrate toward the cathode. As they move through the gel,
longer fragments are retarded more than shorter fragments. As the DNA is negatively charged
do it moves towards the positive electrode. This relationship holds over a range of DNA sizes,
and useful separation can be achieved for DNA fragments from a few nucleotides using a range
of gels of different concentration.

DNA extraction
For DNA extraction first of all splash seat and instruments that are being used in DNA check.
Likewise use gloves and place the equipment’s on their
proper place. Furthermore, prepare buffer .Warming
square ought to had been set at 56 centigrade and look for
protocol. Utilize never-ending marker to name the tested
cylinders. Mix the blood design in tube multiple times to
verify that this is homogenous example. Name the twist
section furthermore and rehash the interaction for residual
examples. On the stop all filtrate must be inside the
assortment tube. Then shift the example to turn tube then
series cylinder can be vacant. In each sample trade tip.
After centrifugation all the sample should be in design tube in the event that indistinguishable
material is in turn, do again centrifugation. DNA example may be at rear inside the cylinder.
While the fixation and the two 6280 proportions are urgent. Likewise two 6230 proportions are
right and those proportions need in the middle between 1.5 to a couple. ..

Western blotting

Next video was about western blotting it is often used in research to separate and identify
proteins. In this video a mixture of proteins is
separated based on molecular weight, and thus by
type, through gel electrophoresis. there are five
steps are involved in western blotting procedure and
detection assay, namely, transfer, blocking, primary
antibody incubation, secondary antibody incubation
and protein detection, and western blotting analysis
for this purpose we use the blotting buffer, gel is
removed by using opening keys and equablaster the
gel with blotting buffer for 15min on a rotating
platform.
We
sandwich
the
fiberpad,blotting paper ,gel (cellulose)by placing
blotting paper over the fiberpad.we perform
gelelectrophresis like putting gel inside it and cooling
furthermore filling it with the blotting buffer. These
results are then transferred to a membrane producing
a band for each protein. The membrane is then
incubated with labels antibodies specific to the protein
of interest The unbound antibody is washed off leaving only the bound antibody to the protein of
interest. The bound antibodies are then detected by developing the film. As the antibodies only
bind to the protein of interest, only one band should be visible (The source of antibody is only
the B OR T immune cells ) The thickness of the band corresponds to the amount of protein
present; thus doing a standard can indicate the amount of protein present.

REAL-TIME PCR:
Quantitative PCR also called real-time PCR is a
PCR-based technique that couples amplification of
a target DNA sequence with quantification of the
concentration of that DNA species in the reaction.
This empowers the scientists to evaluate how
much DNA in the example toward the beginning of
the response.
Working of QPCR
Ongoing PCR has similar standards as normal PCR.
DNA amplificatin continuously "probes" are added to the response.
Probe:
A probe is an oligonucleotide that is named with a fluorescent label and a quencher.
The quencher diminishes the fluorescence power.
During the PCR cycle, the tests denature and toughen to the objective grouping. A fluorescence
columnist is let out of the test for each intensification of the objective succession.
To best see constant PCR, we really want to learn essential PCR.
Ongoing PCR comprises of two significant parts,
1. Thermal cycler
2. Optical module
Thermal cycle has three essential steps of continuously real time PCR
1. DENATURATION
2. ANNEALING
3. ELONGATION
The columnist particle is possibly delivered when TAQ is totally polymerized by the DNA strand.
B. Optical module
This part estimates the fluorescence force in tubes.
Cycle after cycle:DNA amplification increase with the increase in fluorescence of 30 cycles
which plots an amplification graph curve given below

SDS PAGE
SDS PAGE or Sodium Dodecyl
Sulphate- Polyacrylamide Gel
Electrophoresis is a technique used
for the separation of proteins based on their molecular weight. It is a technique widely used in
forensics, genetics, biotechnology and molecular biology to separate the protein molecules
based on their electrophoretic mobility.
Materials Required
Power Supplies: It is used to convert the AC current to DC current.
Gels: These are either prepared in the laboratory or precast gels are purchased from the
market.
Electrophoresis Chambers: The chambers that can fit the SDS-PAGE gels should be used.
Protein Samples: The protein is diluted using SDS-PAGE sample buffer and boiled for 10
minutes. A reducing agent such as dithiothreitol or 2-mercaptoethanol is also added to reduce
the disulfide linkages to prevent any tertiary protein folding.
Running Buffer: The protein samples loaded on the gel are run in SDS-PAGE running buffer.
Staining and Destaining Buffer: The gel is stained with Coomassie Stain Solution. The gel is
then destained with the destaining solution. Protein bands are then visible under naked eyes.
Protein Ladder: A reference protein ladder is used to determine the location of the protein of
interest, based on the molecular size.
Principle of SDS-PAGE
The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the
opposite sign when placed in an electric field. The separation of the charged molecules
depends upon the relative mobility of charged species.
The smaller molecules migrate faster due to less resistance during electrophoresis. The
structure and the charge of the proteins also influence the rate of migration. Sodium dodecyl
sulphate and polyacrylamide eliminate the influence of structure and charge of the proteins, and
the proteins are separated based on the length of the polypeptide chain.
Role of SDS in SDS-PAGE
SDS is a detergent present in the SDS-PAGE sample buffer. SDS along with some reducing
agents function to break the disulphide bonds of proteins disrupting the tertiary structure of
proteins.

REFERNCES

https://www.youtube.com/watch?v=QGX490kuKjg

https://www.youtube.com/watch?v=vq759wKCCUQ

https://www.youtube.com/watch?v=YnF1b_Kqf88

https://www.labxchange.org/library/items/lb:LabXchange:21553aac:video:1

https://www.youtube.com/watch?v=JNl1kjw9ZDQ (https://www.youtube.com/watch?
v=gmNw6CWtN5k

https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html https://www.youtube.com/watch?
v=VgAuZ6dBOfs

https://www.youtube.com/watch?v=uKeMiAZ8Zu4
https://www.youtube.com/watch?v=HmkNzMrhIFQ (