anam siddique gel electrophoresis

Presented by :Anam Siddique

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04/12/2023 02:00:18 AM Agarose Gel electrophoresis and

southern blotting

Agarose Gel electrophoresis and Southern blotting

• ContentsIntroduction of electrophoresisTypes of gel electrophoresisPrincipleProcedureApplicationsAdvantages & disadvantagesSouthern blottingReferences04/12/2023 02:00:18 AM

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Introduction

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Electrophoresis“The movement of

suspended particles through a medium under the action of an electromotive force applied to electrodes in contact with the suspension”

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Gel electrophores

is A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form (like gelatin).

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Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied. Charged particles can include DNA, amino acids, polypeptides, etc.

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Types of gels used in electrophoresis

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Gel

AgarosePolyacrylam

ide

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AgarosePolysaccharideUsed at concentrations of 0.5 to 2%The higher the agarose concentration the "stiffer" the gelUsed for the separation of DNA fragments

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PolyacrylamidePolyacrylamide gel electrophoresis (PAGE)Used to separate proteinsIt is non toxic however un polymerized acrylamide is neurotoxin

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Agarose Polyacrylamide

Larger 'pores' Large particles can

move more easily Used for the

electrophoresis of large molecules such as DNA and RNA

Agarose gels have a large range of separation, but relatively low resolving power

Small poresLarge particles

cannot move easilyUsed for

electrophoresis of small molecules such as proteins

Polyacrylamide gels have a rather small range of separation, but very high resolving power

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Principle

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The agarose gel after solidification has some gaps called PORES.These pores are the channels through which charged molecule has to travel.When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge.Nucleic acids have negative charge due to their phosphate backbone.The migration flow is determined solely by the molecular weight.small weight molecules migrate faster than larger ones.

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buffer

Cathode(negative)

Anode(positive)

wells

DNA

All bands are DNA fragmentsBand 1 longest fragment so nearest to well

It moved slowest.

Band 10 is shortest and moved fast.

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Method

DNA sample AgaroseEthidium bromide6X sample loading dyeDNA ladder standardPower supplyGel casting tray & combPipette and tipsElectrophoresis chamberBuffers Transilluminator

The movement of suspended particles through a medium under the action of an electromotive force applied to electrodes in contact with the suspension”

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Materials required

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1. Dissolve agar in buffer

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2. Add ethidium bromide (1µl) in the agarose gel.

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3. Pour the gel in casting tray

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4. Insert the comb and let the gel solidify

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5. After solidification of gel, carefully remove the comb and place the gel in the gel rig with the wells closest to the cathode (black) end. Cover the gel with 1X TAE running buffer.

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6. Keeping DNA samples on ice, pipette up 5 µl of a sample, add it to one drop (1μl) of loading dye.

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7. Load the mixture into a well

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8. Place the cover on the gel rig and run the samples towards the anode (red) end. And run it at 120 volts for about half an hour.

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9. Turn off the power pack, remove the gel and visualize with transilluminator or U.V. light.

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10. Take a photograph with a polaroid Photo documentation camera.

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Functions of materials

Ethidium bromide

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is used for stainingIt will bind with DNA and & it will emitt fluoresencemutagenic and should be handled with extreme caution

Loading dye

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Gives colour and density to the samplethe dyes are negatively charged and thus move in the same direction as the DNABromophenol blue for dyingGlycerol for density

DNA ladder standard

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useful for quantifying the amount of DNA in your sample bands by comparison with the marker bands

Tank buffers

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TAE buffer (Tris Acetate EDTA) TBE buffer (Tris borate buffer)sequesters divalent cations

TAE buffer

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TAE offers the best resolution for larger DNATAE requires a lower voltage and more timelinear, double stranded DNA runs faster in TAE.

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1X TAE Buffer:1. Trisma base = 242g2. Glacial acetic acid = 57.1ml3. 0.5M EDTA = 100mlMix all the constituents and

raise thevolume up to 1L with distilled

water.

TBE buffer

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TBE buffer  (Tris/Borate/EDTA) is often used for smaller DNA fragments (ie less than 500bp)

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VisualizingDNA

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Ethidium Bromide binds to DNAFluorescence under UV light makes bands visible.

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Results

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Unique bands and or banding patterns.Molecular weight standards can be ran with samples to estimate sizes..Migration or separation of a sample can be measured (Rf values).

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Factors affecting

Gel electrophore

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Voltage usedRunning timeAmount of DNA usedReversal of polarity

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Applications

It can be used todetermine the sequence of nitrogen bases. the size of an insertion or deletion the presence of a point mutation to distinguish between variable sized alleles at a single locus to assess the quality and quantity of DNA present in a sampleDNA Sequencing

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Blotting (Southern Blot)Size of DNA can be determined by comparing it with ladder.Medical ResearchForensics (Criminal investigations) like Blood, Saliva, Hair, nail Paternity Tests

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Advantages and disadvantages

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Advantages Cheap QuickEasySample recovery

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Disadvantage

Toxicity DNA visualization uses dyes like Ethidium Bromide which chelate DNA and is a known carcinogen. Although some safer alternatives are becoming available (SYBR Safe).

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Southern Blotting

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Definition A technique which allows the detection of a specific DNA sequence (gene or other) in a large, complex sample of DNA.

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Principle Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size separated DNA to a filter membrane for probe hybridization.

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ProcedureDigestion + gel electrophoresisDNA is transferred to a sheet of special blotting paperLabel with specific DNA probeDetected on the Xray film

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ApplicationsTo identify a single gene among thousands of fragments of DNA and to detect sequences of DNA in an organism’s genome.Used in gene discovery and gene mapping.To analyze the genetic patterns in an organism’s DNA.To identify gene mutations, deletions, duplications, and gene rearrangements involved in diseases.To determine the number of copies of a particular DNA sequence presented in the genome of an organism.

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To identify related DNA sequences in the genome and to determine if there is a gene family (a group of similar genes.To detect certain cancers and genetic diseases, such as: 

Monoclonal leukemia populationsSickle cell mutations

Used in DNA fingerprinting, genetic engineering, & forensic science for tests such as

Paternity testingSex determination

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References

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• http://en.wikipedia.org/wiki/Gel_electrophoresis • http://www.methodbook.net/dna/agarogel.html• http://abe.leeward.hawaii.edu/Protocols/DNA%20Gel

%20Preparation%20and%20Electrophoresis.htm• http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/

gels/principles.html• http://www.academia.edu/1546698/Gel_Electrophoresis_-

_Principles_and_Basics• www.biotecharticles.com/.../Agarose-Gel-DNA-

Electrophoresis Applications• www.molecularstation.com/agarose-gel-electrophoresis• http://biology.arizona.edu/sciconn/lessons2/vuturo/vuturo/

gel.htm

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Animations• http://www.dnalc.org/resources/animations/gelelectrophoresis.html

• http://learn.genetics.utah.edu/content/labs/gel/

• http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html

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