Richa Parashar is a 4th year student studying Biotechnology. Her document outlines various molecular biology techniques including isolating genomic DNA from bacteria, isolating plasmid DNA, performing agarose gel electrophoresis, restriction enzyme digestion, ligation, PCR, isolating RNA from plants, and synthesizing cDNA using RT-PCR. It provides detailed steps for each technique including required reagents and optimal conditions.
Richa Parashar is a 4th year student studying Biotechnology. Her document outlines various molecular biology techniques including isolating genomic DNA from bacteria, isolating plasmid DNA, performing agarose gel electrophoresis, restriction enzyme digestion, ligation, PCR, isolating RNA from plants, and synthesizing cDNA using RT-PCR. It provides detailed steps for each technique including required reagents and optimal conditions.
Richa Parashar is a 4th year student studying Biotechnology. Her document outlines various molecular biology techniques including isolating genomic DNA from bacteria, isolating plasmid DNA, performing agarose gel electrophoresis, restriction enzyme digestion, ligation, PCR, isolating RNA from plants, and synthesizing cDNA using RT-PCR. It provides detailed steps for each technique including required reagents and optimal conditions.
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.