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Mol Bio Notes

The document outlines various molecular biology techniques including competent cell preparation, bacterial transformation, plasmid isolation, DNA estimation, agarose gel electrophoresis, restriction digestion, genomic and RNA isolation, PCR, and Southern blotting. Each technique includes a detailed process description, highlighting the reagents and steps involved. These methods are essential for manipulating and analyzing DNA and RNA in research and biotechnology.

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15 views1 page

Mol Bio Notes

The document outlines various molecular biology techniques including competent cell preparation, bacterial transformation, plasmid isolation, DNA estimation, agarose gel electrophoresis, restriction digestion, genomic and RNA isolation, PCR, and Southern blotting. Each technique includes a detailed process description, highlighting the reagents and steps involved. These methods are essential for manipulating and analyzing DNA and RNA in research and biotechnology.

Uploaded by

trijitrana9878
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Competent Cell Preparation: Process by which microorganisms accept foreign DNA -> Altered Cell walls; Treatment

of cells with Ca2+ or Mg2+ or electroporation or heat shock


Process: Culture cell in LB media -> When OD600 > 0.3, put cells in ice -> Spin -> Add chilled CaCl2 or MgCl2 -> Spin ->
Decant -> Add chilled CaCl2 or MgCl2 -> Spin -> Decant -> Rinse with ddH2O -> Freeze

Bacterial transformation: Process by which foreign DNA is introduced into cell


Process: Thaw frozen competent cells -> add DNA -> incubate -> heat shock/ electroporation -> put in ice -> add in LB
or SOC media -> incubate

Plasmid isolation:
Process: Culture cell in LB media -> centrifuge -> bacterial pellet -> add Resuspension solution [Tris-Cl (buffer) + EDTA
(disrupts outer membrane by chelating divalent ions and inhibiting DNases)] + Lysis solution [NaOH (lysis of bacteria
and denaturation of DNA+RNA+Protein) + SDS (detergent solubilizes phospholipids and proteins of the cell
membrane resulting in cell lysis and release of its contents)] + Neutralization solution [Potassium acetate (neutralize
+ renaturation of DNA)] -> centrifuge -> remove supernatant and add 70% EtOH -> dissolve in RNase -> Ag Gel
electrophoresis

DNA estimation: A260/A280 ratio

Agarose Gel Electrophoresis: Separates DNA fragments based on their molecular weight using an electric field
(visualization using Ethidium Bromide), Agarose gels are made of (0.7-2% agarose)
Prepare agarose gel -> add loading buffer to each DNA sample -> place agarose gel in gel box -> let it solidify -> fill gel
box with buffer -> add markers and ladder -> run gel at 80-150 V -> turn OFF and remove gel -> observe under UV
light

Restriction digestion: Restriction enzymes cleave DNA at specific sequences, however cell’s own DNA is not cleaved
from these restriction enzymes due to DNMTs/ methylation.
Process: Select restriction enzyme -> add enzyme + DNA + buffer + dH2O -> mix by pipetting -> incubate -> gel
electrophoresis

Genomic DNA isolation: Cell membranes destroyed using SDS, on disruption, endogenous nucleases cause extensive
hydrolysis, however DNA is protected by EDTA as it chelates Mg2+, proteinase K degrades proteins, phenol
chloroform method (DNA in aqueous phase, denatured proteins in interphase, others in organic phase) [Phase
division due to hydrophobicity/ hydrophilicity of nucleotides] [Ph:Chlf:IsoAmylAlco = 25:24:1]
Process: Culture cell in LB media -> centrifuge -> bacterial pellet -> add Resuspension solution [Tris-Cl (buffer) + EDTA
(disrupts outer membrane by chelating divalent ions and inhibiting DNases) + Proteinase K] -> Add tris buffered
phenol-chloroform -> shake -> spin -> decant top aqueous layer -> add phenol chloroform -> add RNases ->
precipitate DNA by adding sodium acetate and 95% isoamyl alcohol -> wash with 70% ethanol

RNA isolation: Guanidinium thiocyanate is a chaotropic agent which can denature proteins, DNases and RNases and
helps in extraction of DNA-RNA
Process: Take cells -> add sodium acetate pH 4 -> add phenol + chloroform + isoamyl alchohol -> store in ice ->
centrifuge -> pipette aqueous phase -> add isoamyl alcohol -> add denaturing solution (guanidinium thiocyanate +
sodium citrate + N-laurosylsarcosine + mercaptathenol) -> resuspend RNA pellet in 70% ethanol -> centrifuge -> air
dry -> dissolve RNA pellet in DEPC (diethyl pyrocarbonate-used as RNase inhibitor) treated water

PCR: Amplify segments of DNA of interest by denaturation of whole template DNA (94OC), annealing of
oligonucleotide primers to single stranded target sequences (54OC) and extension of annealed primers by
thermostable DNA polymerase (72OC)
Process: Add reagents (deionized water + Polymerase + buffer + dNTPs + both primers + template DNA) -> switch ON
thermocycler -> analyse by gel electrophoresis

Southern Blotting: Locate a particular sequence of DNA by gel electrophoresis followed by labelled probe
hybridization
Process: isolation of DNA -> restriction digestion -> agarose gel electrophoresis -> electrophoresed DNA from
electrophoresis gel to nitrocellulose membrane inside transfer buffer -> blocking free surface of membrane ->
hybridization using probe buffer -> incubate -> check fluorescence

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