Report 2: Genetics Laboratory

INTERNATIONAL UNIVERSITY- VIETNAM NATIONAL UNIVERSITY
GENETICS LABORATORY
REPORT 2
GROUP : 3
DATE : 25/04/2019.
MEMBERS OF GROUP:
- Trần Thị Vân Anh – BTARIU17031
- Phạm Quốc Tính – BTBTIU17150
- Hà Nghị Phong – BTBCIU17028
- Phan Minh Tuyền – BTBTIU17151
1)Explain the function of SDS buffer? Can we use SDS without grinding in
DNA extraction protocol ? Explain why?
- SDS stands for 'sodium dodecyl sulfate' is a solid anionic detergent that can solubilize the proteins
and lipids that form the membranes. It removes the negative ions from the protein and destroys its
confirmation. Because of loss of confirmation the protein loses its structure. The proteins from the
cell membrane get damaged and cell gets broken. This will help the cell membranes and nuclear
envelopes to break down and expose the chromosomes that contain the DNA. In addition, SDS
helps release the DNA from histones and other DNA binding proteins by denaturing them.
- We can not use SDS without grinding in DNA extraction protocol. This is because the plants
have DNA, RNA and proteins, we can not do experiment without grinding . When grinding in
DNA extraction protocol, the process in removing impurities such as proteins and RNA from the
sample is easier.
2)It is always recommended that organic extraction step should be repeated
three times. Explain why?
In a multiple extraction procedure, a quantity of solvent is used to extract one layer (often the
aqueous layer) multiple times in succession. The extraction is repeated two to three times, or
perhaps more times if the compound has a low partition coefficient in the organic solvent.
In a multiple extraction of an aqueous layer, the first extraction is procedurally identical to a single
extraction. In the second extraction, the aqueous layer from the first extraction is returned to the
separatory funnel,with the goal of extracting additional compound. Since the organic layer from
the first extraction had already reached equilibrium with the aqueous layer, it would do little good
to return it to the separatory funnel and expose it to the aqueous layer again. Instead, fresh diethyl
ether is added to the aqueous layer, since it has the potential to extract more compound.
The process is often repeated with a third extraction , with the aqueous layer from the second
extraction being returned to the separatory funnel, followed by another portion of fresh organic
solvent. In multiple extractions, the organic layers are combined together,as the goal is to extract
the compound into the organic solvent.
When an aqueous solution is extracted with an organic solvent that is denser than water (for
example dichloromethane, CH2Cl2CH2Cl2), the only procedural difference is that there is no need
to ever drain the aqueous layer from the separatory funnel. After draining the organic layer from
the first extraction, fresh solvent can be added to the aqueous layer remaining in the funnel to begin
the second extraction .
3)Function of ammonium acetate? Can we replace this salt by other salt?

Function of ammonium acetate in DNA extraction is remove cellular and histone proteins bound
to the DNA by having precipitated the protein. In addition, absolute ethanol can cause harm to
DNA in precipitation process, so adding ammonium acetate help DNA renaturation.
Yes, we can replace ammonium acetate by other salts but we must change method.
4) Why do you have to use absolute ethanol for precipitation and ethanol 70%
for washing?
- DNA is insoluble in ethanol. We use absolute ethanol for precipitation in order to get good
amount of DNA .
-Washing the precipitate with 70% Ethanol is to remove the excess of salts from the DNA (i.e. the
excess of salts dissolve in the 30% of water). Because of the concentration of ethanol is low, it do not
cause harm to the DNA.
5) Which methods to qualify and quantify DNA that you know?

What is (are) advantage(s) of agarose gel electrophoresis?
+The main purpose of agarose gel is to isolate and analyze the DNA molecules which are cut by
restriction enzymes. These cut pieces of DNA can be used for the cloning purpose to make varius
plasmids from one fragment. The function of gel electrophoresis is to isolate cut plasmids or vector
from the uncut vectors
+Agarose gel can also be used to separate the RNA or protein molecules form the gel. DNA
fragment re separate before Southern blotting and RNA and proteins are separated before the
Northern blotting. It also allows the DNA fragment to pass through PCR to make various copies
of DNA molecules, so that they can be used in DNA fingerprinting or in any other method. DNA
molecules of same size can be isolated through the agarose gel, when the electric current is passed
through it.
+Quantify and quatify of the size DNA molecules is analyzed by lambda DNA ladder and by
observing the absence of streaking of fragments respectively. It means that only those DNA
fragments can be observed or isolated which have fluorescent streak in them so that they can be
identified.
+There are many benefits to using precast agarose gels. The principal advantages of precast gels
include both speed and the assurance of consistent run conditions across loadings and between
experiments. This consistency is particularly useful when screening for small changes or
comparing a large number of samples.
+Ready Agarose™ precast agarose gels come in ready-to-load UV-transparent running trays with
fluorescent well numbering, a handy feature for sample identification with a large number of
samples or a complicated loading pattern. The trays also have a fluorescent ruler along one side
for convenient measurement of band migration.
+The locking tray system prevents the loss of samples from wells, and bands remain sharp because
all the samples remain confined within the wells prior to electrophoresis down the gel.
6)You should always prepare agarose gel and run electrophoresis in buffer.
Why?
- EDTA has some important role to play in this combination. EDTA is a chelating agent. It chelates
the Mg2+ ion which is required for enzyme DNAse as a cofactor. DNAse is an enzyme which
cleaves DNA into small fragments.
- So by addition of EDTA, our DNA is protected from the enzymatic activity. Further, the buffer
will neutralize the charge of a water molecule.
- Under the electrical current, the water molecule dissociates into H+ and OH-. The H+ ions can
react with the PO3- of DNA. So the negative charge of buffer neutralize the charge of the water
and protects DNA.
- TAE has a lower buffering capacity while TBE has a higher buffering capacity as compared to
TAE buffer. However, the borate can react with the sugar backbone of DNA, therefore, it is not
always recommended.
- Further, if glycerol is a component of your DNA gel loading dye, then TBE can be a major set
back. The borate reacts with the glycerol and decreases the activity of DNA loading dye.
- Hence if glycerol is the primary component in the DNA gel loading dye, TAE buffer is the best
choice for getting a good result.
7) Show agarose gel picture in your report ? Do you find DNA in your sample
? Is it good? Explain how good it is?
- We can not find DNA in our sample . This is because smears happens above the well head . This
indicates that the DNA is contaminated with salt due to salt remaining in the DNA washing
process with ethanol 70%.
- In addition, there are long traces because the DNA is broken by the DNA extraction process. The
bright smear at the bottom is RNA because RNA has a smaller molecular weight than DNA, so it
runs faster than DNA

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INTERNATIONAL UNIVERSITY- VIETNAM NATIONAL UNIVERSITY

3)Function of ammonium acetate? Can we replace this salt by other salt?

5) Which methods to qualify and quantify DNA that you know?

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