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Applied science, biology, unit 11D
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- PEARSON (PEARSON)
this is biology- unit 11 D - genetic and genetic engineering This is distinction level by teacher.
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BiologyCollecting DNA from plants
Equipment : FTA® Plant Card, piece of plant material, pestle, pen
Healthy and safety
Lab coat to be worn with safety glasses
hazard warning and safety sign to be read before any work
Bags or coat must be kept under bench or completely out of the way
Spills or accident must be told to a teacher. Rinse skin instantly if any chemical touches
Broken glass must be cautiously place in the yellow glass bin provide
Method
On the FTA® Plant Card, fold back the cover. And place a new leaf on card. Make sure the plant
matter doesn't go past one of the boxes printed onto the card. Secure the cover above the plant
material and pound the leaf into the card with something like a pestle until moisture oozes onto the
card's back. The goal is to stop the specimen from migrating outside of the card's printed box since
doing so could contaminate nearby samples. The plant material should be discarded. On the FTA®
Plant Card's cover, wite the given name of the plant in the relevant "Sample ID" slot. After the plant
material has already been crushed onto the card, the cover should really be labelled since the pestle
can erase the writing from the exterior. Allow the card to dry at while the cover is folded back. A
minimum of an hour at room temperature. Avoid heating the card due to the fact that doing so
could cause PCR inhibitors to adhere to the card.
Theory behind the technique
Filter paper that has been treated with SDS, Tris/EDTA (TE) buffer, and uric acid is called FTA® card.
The plant cells are split open by the detergent SDS. The DNA deteriorate enzymes are unable to
function because EDTA binds to the metal ions needed by the enzymes as co-factors. To guarantee
that the EDTA has the desired chelating effect, tris (a weak base) is required. Uric acid safeguards
DNA from damage caused by free radicals.
The filter paper used in the FTA plant card has been treated and impregnated with chemicals
including guanidine salts and detergents. In order to release the DNA for binding to the card surface,
these substances induce the cells to lyse and the proteins to become denatured. When plant tissue
is pressed onto the surface of the card, the DNA from the cells is transferred onto the treated paper
and binds to it, becoming trapped and stabilized.
Cell lysis is the process of releasing a cell's contents by rupturing the cell wall and membrane. In
biology, cell lysis is frequently used to liberate DNA from cells for use in PCR or DNA sequencing.
Detergents, enzymes, sonication, grinding, and other chemical and physical processes can all be used
to lyse cells. These techniques damage the cell membrane and cell wall, allowing DNA and other
cellular components to escape. In the practical, we used a mortar to disrupt the cell wall and
membrane and pass the cellular component to the FTA plant card.
The stability and integrity of the DNA molecules can be impacted by residual moisture in the DNA
sample, which can cause the sample to degrade and produce results that are inaccurate or non-
,specific. Water can also compete with DNA for binding to PCR amplification enzymes like Taq
polymerase, reducing PCR specificity and efficiency.
1. Cell lysis: The opening of the cells to release the DNA is the first step in the DNA extraction
process. This can be done through mechanical interference, chemical lysis, or a combination
of the two. The nuclear envelope and cell membrane are typically destroyed by detergents
during chemical lysis, allowing the DNA to be released.
2. DNA purification: DNA purification is the process of separating the DNA from other cellular
elements like proteins, lipids, and RNA after it has been released from the cells. This is
typically accomplished by lysate, which causes the DNA to precipitate out of solution, by
adding salts and organic solvents. The remaining components can then be filtered or
centrifuged to separate the DNA from them.
3. Quantification of DNA: After purification, it's critical to assess the DNA's quantity and quality.
Multiple methods, such as UV spectrophotometry, fluorometry, or gel electrophoresis, can
be used to accomplish this.
Cleaning the DNA sample
Equipment: cutting board between, punch, 1.5 mL microcentrifuge tube, plastic wire, pen, syringe,
graduated yellow tip, Purification Reagent, µL TE-1 buffer
Healthy and safety
Lab coat to be worn with safety glasses
hazard warning and safety sign to be read before any work
Bags or coat must be kept under bench or completely out of the way
Spills or accident must be told to a teacher. Rinse skin instantly if any chemical touches
Broken glass must be cautiously place in the yellow glass bin provide
Method
Between the dry FTA® Plant Card and the backing, place a cutting board. Put the punch's tip over the
zone that requires to be sampled—a region that is chlorophyll-colored green. To extract a paper
disc, punch it firmly while rotating it. If the extracted specimen intersected with some other sample
in an adjacent box, do not etract the sample as to prevent cross contamination. Use the plastic wire
provided to pry the tiny disc out of the punch and into a clean, colourless 1.5 mL microcentrifuge
tube. The species utilised should be noted on the tube's label. Towards the tube containing the
paper disc, add 100 µL of Purification Reagent using only a 1 mL syringe with a graded yellow tip. To
wash the disc, close the tube and flick Every 30 seconds for two minutes. Purification Reagent should
be taken out of the tube using the same yellow tip previously used. Attempt to get rid of as much of
the froth as you can from the tube. Repeat the processed of using putting purification reagent all
over gain.
, Use a fresh yellow tip connected to a 1 mL syringe and fill the tube with 100 µL TE-1 buffer. To wash
the disc, close the tube and flick Every 30 seconds for two minutes. Discard and eliminate the TE-1
buffer from the tube by employing the same yellow tip. repeat the process of usingTE-1 buffer all
over again
Theory behind the technique
it is essential to purify and remove any contaminants, such as proteins, salts, and other cellular
components, that may interfere with downstream applications. One of the ways to achieve this is by
washing the DNA with a purification reagent. A detergent found in the Purification Reagent aids in
clearing the disc of cell debris, including PCR inhibitors.
In order to resuspension and store DNA, TE-1 buffer, a Tris-EDTA buffer, is frequently used. Tris,
which aids in preserving a stable pH, and EDTA, which chelates metal ions that may damage DNA,
are both present in TE-1 buffer. Additionally, there are no salts in the buffer, which prevents them
from interfering with applications used later. It is also used to wash away the purification reagent
that might be present in the DNA sample. The puncher and a plastic wire is used to prevent
contamination in order to prevent contamination
Amplification of the DNA
Equipment : 0.5 mL microcentrifuge tube, microsyringe, , white graduated tip, water bath, distilled
water, primer 1 and 2 , pen, FTA disc, foam floater.
Healthy and safety
Lab coat to be worn with safety glasses
hazard warning and safety sign to be read before any work
Bags or coat must be kept under bench or completely out of the way
Spills or accident must be told to a teacher. Rinse skin instantly if any chemical touches
Broken glass must be cautiously place in the yellow glass bin provide
Method
Add the following to a 0.5 mL microcentrifuge tube containing a PCR bead using a microsyringe fitted
with an clean, white, graduated tip for each reagent:
– 10 µL of Primer 1
– 10 µL of Primer 2
– 4 µL of distilled water
Shut the tube. To combine the ingredients and hasten the PCR bead's dissolution, gently flick the
tube's bottom. To gather the liquid at the tube's bottom, firmly tap the tube against the bench. A
pair of forceps should be used to insert the FTA® disc created in "practical 2" into the tube
containing the PCR reagents. Make certain that the disc is immersed liquid in the tube. Affix your
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