NAME- Richa Parashar

BRANCH- Biotechnology (4th year)

ROLL NO.- 1614354045
 Molecular Biology & R-DNA
Technology
 Laboratory skills in Molecular Biology
 Isolation of genomic DNA from bacterial cells
 Isolation of plasmid DNA
 Agarose gel electrophoresis of DNA
 Restriction Enzyme Digestion
 Ligation
 Preparation of Competent Cells
 Transformation & blue white selection
 Polymerase Chain Reaction
 Isolation of RNA from Plant Sample
 Cdna synthesis using RT-PCR
 To Separate and Visualize DNA by Agarose Gel
Electrophoresis
 Agarose gel electrophoresis is a powerful and widely used method
that separates molecules on the basis of electrical charge, and size.
 Used to separate DNA, RNA and protein.
 Condition of charge, size and shape interact with one another
depending on the structure and composition of the molecule, buffer
condition, gel thickness and voltage.
 They are made with conc. Ranging between 0.7%(provides good
resolution of 5-10Kb fragments) and 2%(good resolution for small
0.2-1Kb fragments)
 Ethidium bromide(EtBr), a chromogen, is added to the gel to
visualize the separate DNA under UV transillumination .
To Isolate the Genomic DNA from Bacterial cells
 The isolation and purification of DNA from cells is one of the most
common procedure in contemporary biology.
 SDS- sodium dodecy sulphate , this is the reagent used to distrupt
the cell membrane.
 DNA can be protected from endogenous nucleases by chelating
Mg2+ ions using EDTA.
 Phenol and Proteinase enzyme is used to degrade the proteins in
the disrupted cell soup.
 Phenol and Chloroform are used to denature and separate proteins
from DNA.
To Isolate Plasmid DNA from Bacterial Cells
 . PLASMID- Prokaryotic, double standed circular DNA molecule
 Plasmid are widely used cloning vehicles which are replicons that
are stably inherited in an extra-chromosomal state, ranges around 1-
200 Kb.
 Isolation of plasmid DNA from chromosomal DNA in many ways like
NaOH is added to the solution which denatures the DNA in to small
single strands.
 Also potassium acetate to precipitate the chromosomal DNA, and
proteins along with the other debris
 The denature proteins form a layer at the interface.
 The plasmid DNA in the aqueous phase is precipitated with ice cold
ethanol or isopropanol.
To Digest the DNA with Restriction Enzyme
 Restriction digestion is a process of cutting DNA molecules in to
smaller pieces with special enzymes called restriction endonuclease
which has a specific sequence to recognize.
 The most abundantly used restriction enzymes are type II restriction
enzymes.
 NaCl and MgCl are required for the activity of restriction enzymes.
 They basically cleaves the phosphodiester bond in between the
specific bases, one on each DNA strand.
 The restriction endonucleases produce either sticky or blunt ends
upon cleavage.
 Also based on the number of sequences identified for cleavage
restriction enzymes can be tetracutter (4), hexacutter (6), or
octacutter(8).
To Ligate the linearized Plasmid DNA and the Insert
DNA
 Ligation is the process of joining of two fragments of nucleic acid by
using enzyme ligase.
 Ligase catalyses the formation of phosphodiester bonds between
the directly adjacent 3’ hydroxyl and 5’phosphoryl termini if nucleic
acid molecule.
 The requirements are : ATP dependent, optimum conc. Of salts and
phosphate, incubation time, and temperature(2-4 hrs at 16* C for
sticky ends and overnight at 4* C for blunt ends).
 T4 dna ligase is been used and the source is T4 Bacteriophage
To Prepare Competent Cells of E.coli DH5a by
TSS(transformation and storage solution) Method

 methods are; PEG(polyethylene glycol) based used for both

bacterial as well as yeast.
 This method is technically easy ,simple and yields transformants
with an efficiency of10^6 – 10^7 transformants DNA.
 ELECTROPORATION; (brief exposure of cells to an electric field)
 CaCl2; obtained by creating pores in bacterial cells by suspending
them in a solution containing high conc. If calcium. DNA can then
forced into the host cell by heat shock treatment at 42 C .
 Reagents of TSS solution;
 PEG (polyethylene glycol); fusing agent
 DMSO(dimethyl sulfoxide) a reagent that prevents cytopasmic
water to crystalize.
 MgCl2.H2O
 LB Medium.
To Amplify the given DNA using Thermocycler(PCR)

 This technique is developed by Kary Mullis in 1983.

 PCR is used to amplify specific regions of a DNA stand.
 Most PCR methods typically amplify DNA fragments of upto 10 Kb.
 The general requirements of PCR are:
 DNA template that contains DNA region to be amplified.
 Taq polymerase to amplify the DNA.
 Deoxynucleoside triphosphate (d NTP’s)
 Two primers, which are complementary to the DNA regions at the 5’
and 3’ ends of the DNA regions.
 Buffer solution
.
To Isolate RNA from Plant Sample Using Trizol
Solution
 Trizol is a acidic soln. contains GITC(guanidinium Iso ThioCyanate)
and Phenol
 GITC denatures the proteins and RNase.
 Addition of chloroform after centrifugation separate the soln. into
aqueous and organic phases and RNA remains only in in the
aqueous phase.
 Then RNA can be precipitated with isopropyl alcohol.
 RNase enzyme must be inactivated by using
diethylpyrocarbonate(DEPC)
 This RNA can be used in Cdna synthesis, Northern blot analysis, in
vitro translation, poly (A) selection, RNase protection assay, and
molecular clonning.
To synthesize Cdna using RNA as template by RT-
PCR
 DNA which is synthesise from a messanger RNA using the enzyme
Reverse transcriptase(RTs) is known as c DNA.
 RTs use an RNA template and a short primer complementary to the
3’ end of the RNA to direct the first strand c DNA .
 The second complementary strand can be synthesise by using PCR
 Thus combination of RT and PCR (RT-PCR) allows the detection of
low abundance RNA in a sample.
 It also facilitate the cloning of low copy gene.
 Alternatively the first strand c DNA can be made double stranded
using DNA Polymerase 1 and DNA Ligase.