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Agarose Gel
Electrophoresis of DNA
Mejico, Aia
Melchor, Kevin
Mendoza, Angelo
Mendoza, Edrian
Mendoza, Janel

Introduction

Gel Electrophoresis
● Method for separation and analysis of
macromolecules (DNA, RNA and proteins)
and their fragments, based on their size and
charge

● This phenomenon is called sieving.As a general rule… ● Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. .

Separation of DNA Fragments by Gel Electrophoresis •Two Types – Polyacrylamide gels – Agarose gels .

Polyacrylamide Gel ● Separate DNA fragments up to 1000 bp long ● Higher resolving power than agarose ● Separate fragments that differ in length by single nucleotide ● Separating proteins ranging in size from 5 to 2.000 kDa due to the uniform pore size .

Polyacrylamide Gel ● Run in vertical direction ● Used in DNA sequencing ● Traditional DNA sequencing techniques such as Maxam-Gilbert or Sanger methods used polyacrylamide gels to separate DNA fragments differing by a single base-pair in length so the sequence could be read .

but are optimal for electrophoresis of proteins that are larger than 200 kDa .Agarose Gel ● Separate mixtures of fragments up to 20 kb ● Easier to prepare ● Lower resolution ● Agarose gels do not have a uniform pore size.

Agarose Gel ● Entire chromosomes containing millions of nucleotides can be separated on these gels by applying pulsed electric fields in different directions ● Used in DNA separation .

Objectives ● To be able to separate the DNA using Agarose Gel Electrophoresis ● To be able to answer the guide questions for this experiment .

Materials .

Materials Agarose Tris Acetate EDTA buffer Fast Blast DNA Tracking Dye .

Materials Horizontal Gel Apparatus and combs Power Supply Gel Documentation Apparatus Ultra Rocker Rocking Platform .

Procedure .

Procedure A. Assembly of Agarose Gel Electrophoretic Cell .

Procedure B. Agarose Gel Preparation 1% Agarose in 100 ml TAE buffer Solubilize. milky. it should be hard. and opaque . then cool to 50-60 C before pouring in gel holder Well-forming comb is placed in one edge of the gel Combs were removed gently TAE buffer is poured into gel tank completely covering the gel Gel is poured in the holder/chamber. After 20 mins.

Procedure C. Loading of DNA Samples 10 ul of PV92 XC loading dye was added to each PCR tube except MMR (Molecular Mass Ruler) The samples were loaded in the 8 wells using a clean tip per sample .

Procedure Lane Sample Load Volume 1 MMR (DNA Standard) 10 ul 2 Homozygous (+/+) control 10 ul 3 Homozygous (+/-) control 10 ul 4 Heterozygous (+/-) control 10 ul 5 Student 1 20 ul 6 Student 2 20 ul 7 Student 3 20 ul 8 Student 4 20 ul .

electrical leads connected to the power supply Turn on power supply. Loading of DNA Sample Lid was secured on the gel box.Procedure C. set to 100 V and electrophorese for 30 minutes Turn off power after electrophoresis and gently nudge the gel into staining tray .

convenient. and nontoxic alternative to ethidium bromide .For quick visualization of DNA bands in 15 minutes . safe.Procedure D. QUICK Staining of Agarose Gel in 100X Fast Blast DNAStain .

Procedure Stain gels for 2-3 minutes Rinse gels in warm tap water for 10 seconds Visualize DNA in the gel documentation system Wash gels in warm tap water for 5 minutes twice Examine the gels for expected DNA bands. Bands develop 5-15 minutes after 2nd wash .

Results and Discussion .

Results Expected Actual .

Discussion .

Discussion ● detect Alu(300bp) sequence on PV92(641bp) region in chromosome 16 ● Samples with Alu sequence will travel less because of its bigger size .

Sources of Errors ● ● ● ● ● ● Low concentration of loaded sample Prolonged time prior to visualization Well contamination Duration of electrophoresis Improper loading technique Gel problem .

What is the difference between agarose and polysaccharide gel electrophoresis as a separate technique for nucleic acids? Agarose Gel Polysaccharide Gel Non uniform larger pore size Uniform pore size Optimal for proteins larger than 200 kDa Optimal for protein size ranging form 5 – 2000 kDa Separate DNA fragments from 50 bp to several megabases (larger fragments) Separate DNA fragments differing by single bp (smaller fragments) Low resolving power. smaller range of separation . greater range of separation High resolving power.

Gel electrophoresis separates DNA fragments by size in a solid support medium.What is the principle behind the separation of DNA using gel electrophoresis? This technique allows the detection of small differences between related DNA molecules. The rate of migration is proportional to size: smaller fragments move more quickly. and wind up at the bottom of the gel. .

ethidium bromide. Illumination with ultraviolet light causes the intercalated dye to fluoresce with a pale pink colour. DNA fragments take up the dye as they migrate through the gel. Larger fragments fluoresce more intensely .What is the principle behind the separation of DNA using gel electrophoresis? DNA is visualized by including in the gel an intercalating dye.

phosphate molecules (negative charge) Negatively charged DNA molecules-migrate towards the anode (positive pole) .Why will the DNA move toward the anode? DNA backbone .

Guide Questions 4 and 5 .

aromatic ring) . nitrogen-containing.What is the role of ethidium bromide and what precautions would be observed when handling this reagent? Why? lEthidium bromide: intercalating agent ¡Used a nucleic acid stain ¡Fluoresce with a red-orange color ¡Aromatic ¡Tricyclic structure with aniline groups on either side of pyridine (a six.atom.

¡Moves into the hydrophobic environment between the base pairs lAway from the water molecules (water: fluorescent quencher) lRemoval of water: ethidium bromide will fluoresce ¡Strongly mutagenic lIntercalates into double stranded DNA lWear gloves (latex or nitrile rubber gloves) •Laboratory coat and goggles .

When using a UV transilluminator. what safety precaution should also be observed and why? lUsed in visualization of nucleic acids stained with fluorescent dyes such as ethidium bromide and acridine orange in gel electrophoresis lEmits a short wave UV light lIt can cause severe damage with very short exposure periods .

lProlonged exposure to ultraviolet light also causes premature aging and cancer of the skin .Exposure and Hazards of UV lExposure to UV light: a serious threat to both the eye and skin lPhotokeratitis is an inflammation of the cornea (outer protective coating of the eye) that is caused by exposure to ultraviolet radiation lEye injury can occur due to very brief exposure or with just a flash of intense UV lErythema is sunburn of the skin and can occur within a few seconds of exposure to a concentrated form of UV.

and long sleeves lab coats lUse thick gloves such as thick nitrile or latex gloves Never allow the eyes or skin to be exposed to UV light l .Laboratory Safety Precautions Transilluminators are never to be used without the protective shield in place lNever operate without the safety hood or cover in place when capturing gel images lAlways wear protective equipment such as gloves. face shields.