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Practical Biotech MSC

1) The document describes a procedure to determine the molecular weight of linear double stranded DNA fragments using agarose gel electrophoresis and calculating the Rf value. 2) DNA samples are run on an agarose gel and the distance traveled is used to calculate the Rf value. A standard curve of Rf values versus known DNA sizes is generated to determine the molecular weight of test samples. 3) The procedure involves running samples and a DNA ladder on a gel, measuring distances traveled, calculating Rf values, and using the standard curve to determine molecular weights of test samples.

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Er Parshant Saharan
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123 views6 pages

Practical Biotech MSC

1) The document describes a procedure to determine the molecular weight of linear double stranded DNA fragments using agarose gel electrophoresis and calculating the Rf value. 2) DNA samples are run on an agarose gel and the distance traveled is used to calculate the Rf value. A standard curve of Rf values versus known DNA sizes is generated to determine the molecular weight of test samples. 3) The procedure involves running samples and a DNA ladder on a gel, measuring distances traveled, calculating Rf values, and using the standard curve to determine molecular weights of test samples.

Uploaded by

Er Parshant Saharan
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Aim: To determine the molecular weight of linear double standard DNA

fragments, by Rf value.

Principle: Agarose get electro phases is a commonly used technique for


resolving nucleic acids.
can also be used to determine the size of linear double strong DNA molecule
by comparing its electrophonic
mobility with that of standard molecular weight DNA marker.
The value Rf is defined as the distance travelled by a bond divided
by the
distance travelled by the tracing dye. The relationship between the logarithm
of the relative molecular of each bond
of standard DNA marker and its Rf value is flitted using this standard curve
molecular size of test samples can be determining.

Material Required:

Equipment: Rocker (Optional)

Glassware: Beaker, conical flask, measuring cylinder, staining trey

Regent: Distilled water

Other Requirement: Micropipette, Tips


Procedure :
1) prepare a 1% agarose gel.
2) Load 25ul each of 500 bp ladder and test samples 1,2 and 3 onto the 1%
agarose gel.
3) Electrophorese the gel at 50 volts for 1-2 hours till the bromophond blue
dye reaches the bottom of agarose gel.
4) Measure the distance travelled by the Promophond Blue from the well,
before attaining the gel.
5) Following standing and distance the gel , Visualize the gel against a light
background .
6) Measure the distance in cm travelled by each of the bands of 500 bp ladder
and the test samples of 1, 2 , and 3 from the well.
7) Calculate the rf value using the formula Rf = Distance travelled by the DNA
molecule/Distance travelled by the dye
8) Plot the Rf value each of the bonds of the marker against the corresponding
molecular size to construct a standard graph on a Semi - long graph sheet
9) Calculate the rf value of test sample and obtain the corresponding molecular
size from the graph
For eg: If distance travelled by the dye 8.4 cm & following are the RF
Value obtained for the marker & test samples.
Aim : To learn Plasmid Preparation by alkaline lysis method

Principle: Plasmids are double standard, circular self-replicating extra


chromosomal DNA molecules
They are commonly used to isolate plasmid DNA essentially involving the
following steps:

Growth
Harvest and lysis of bacteria
isolation of plasmid DNA
Alkaline lysis method for rapid purification of plasmids expose the pontifical
difference
between plasmid circles and linear chromosomal fragment cells are used by
the treating with alkaline <NAHO> and a detergent sodium dodecyl sulphate
<SDS>. SDS denature bacterial proteins and NAHO denature the plasmid and
Chromosomal DNA However in the case of plasmids the stands remain closely
circularize since they are linked by the intertwined backbones od double helix.
In contrast stands of linear / nicked
DNA are free to separate completely. When this mixture of denatured plasmid
and chromosomal DNA is neutralized by the addition of plasmid is rapid and
as=curate
since the strode are already in close physical proximity. Linear molecules
generated by the random shearing of chromosomal DNA renature less
accurately forming networks of DNA that Can be
Removed from lysate by Centrifugation, together with Denatured Protein and
DNA remains in Solution and can participle using ethandlise propanol
Agarose gel electrophoresis separates plasmid DNA by its size and
shape. A preparation of Plasmid DNA has Predominately Supercoiled from that
runs faster by virtue of its compact structure, introduction of single / double
stands breaks lead to presence of relaxed and linear from respectively. shade
forms run more slowly
since their open structure experiences more resistance phasing through the gel
matrix. these are seen as bonds above the supercoiled from. however, the
presence of linear form is very rarely seen in a plasmid preparation

Material Required:

Requirements: Gel Roker ( Optional ) Incubator , Shaker , Micro centrifuge ,


Vortex Mixer

Glassware: Beaker , conical flask , Test tubes , measuring cylinders , Petri


plates , Standing trey

Reagent : Distilled water

Other Requirements: Crushed ice , Micropipettes. Tips


Procedure :
Day 1 : Revival of host:-
1) Break open the lyophilized vial and resuspend the sample by adding 0.1
ml of LB brath.
2) Streak a locpful of this suspension on to two LB plates containing
ampicillin at a concertation of 100 ug/ml.
3) Incubate the plates at 37 digree c. , overnight.

Day2:
4) Pick a single cdony from the LB plates and inoculate into 10ml LB broth
containing ampicillin <100ug/ml>
5) Incubate in a shaker set at 37 degree centigrade

Day 3 :
6) Pipette 1.5ml culture each into 5 individual 1.5ml vials .
7) Spin at 6000 rpm for 8-10 min. Discard the supernatant and invert the
vial on blotting paper to drain out left over supernatant place on ice.
8) Respond cell pellet in 100 ul of ice cold solution. Vortex gently place on
ice for 5 min and shift to room temperature.
9) Add 200 ul of solution II at RT mix gently by inverting the vial five times.
10) Add 150 ml of solution III . Mix gently by inverting the vial . Place
on ice for 5 minute.
11) Spin at 6000-8000 rpm for 10 minutes.
12) Transfer the supernatant immediately to a fresh vial and add 450
ml of solution 4 to participate the DNA. Mix by inverting the Vial.
Incubate at RT for 10 -15 minutes.
13) Spin at 10000 rpm for 20 minutes or at 6000 rpm for 30 minutes.
Decant the supernatant. Invert the vial on blotting paper to drain out left
over. Supernatant DNA will be seen as white Precipitate, Sticking to the
side of the vial.
14) Dry the samples at 37 degrees Celsius for 15-20 min. till there is
no trace of solution 4.
15) Respond the pellet in 20 ul of 1 x te . mix by tapping the vial so
that DNA goes into solution.
16) Add 5ul of Ranse A to three vials 4 Incubate at 42 degrees for 5
min. or 37 degree for 20 min. label these vials, do not add Rnose A.
17) Meanwhile, Prepare 1% agarose gel.
18) Add 2ul of gel loading buffer to each of the 5 samples.
19) Load 20 ul of extracted DNA < 5 samples > along with 30 ul of
control DNA samples on 1% agarose gel and electrophorese at 100 volts
for 1-2 hours.
20) Strain with 1X staining dye.
21) Distain to visualize the bonds.

Interpretation :

On analysing plasmid DNA after electrophoresis, one observe two bands


corresponding to nicked and supercoiled DNA. As is seen , the supercoiled
from runs faster than the nicked from due to is compact structure.
RNA , Which is a small Molecule , is seen migrating faster the supercoiled DNA
in samples not treated with R Nase A.

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