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Restriction Digestion Restriction Digestion

© © © © © Bangalore Genei, 2007 © © © © © Bangalore Genei, 2007

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GeNeiTM Restriction DigestionTeaching Kit

Manual

Cat No. New Cat No.

KT01 106211

KT01A 106212

KT01B 106213Revision No.: 01010606



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CONTENTS

Page No.

Objective 3

Principle 3

Kit Description 7

Materials Provided 9

Procedure 10

Observation & Interpretation 12

AGAROSE GEL ELECTROPHORESIS

Introduction 15

Principle 15

Procedure 18

ORDERING INFORMATION 20

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Objectives:• To perform restriction digestion of λ DNA with EcoR I

and Hind III enzymes.• To analyze the restriction pattern by agarose gel

electrophoresis.

Principle:Restriction Enzyme Activity:

Bacteria are under constant attack by bacteriophages.To protect themselves, many types of bacteria have developeddefence mechanism in the form of enzymes calledendonucleases that chop up any foreign DNA. Since theseenzymes restrict the infection of bacteriophages they aretermed "restriction endonucleases". These molecularscissors found in the bacterial cytoplasm can provedangerous to the cell, so bacteria protect their own DNA bymethylating the adenine or cytosine bases. The methyl groupsblock the binding of restriction enzymes, but not the normalreading and replication of genetic information. DNA from anattacking bacteriophage will not have these protective methylgroups and will be destroyed.

Together restriction enzyme and its modificationmethyltransferase form a restriction modification (RM)system. Four kinds of RM systems are known. These aredistinguished based on subunit composition, kinds ofsequences recognised and cofactors needed for activity. Mostcharacterised enzymes (about 93%) belong to Type II class.They comprise the commercially available restrictionenzymes used for DNA analysis and other manipulations.

Note: All discussions in the manual pertain to Type IIRestriction enzymes

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Each restriction enzyme has a unique name, derived fromgenus, species and strains of bacteria that produce them,followed by a number that refers to discovery order. e.g.EcoR I and EcoR V are both from Escherichia coli, strain R;I and V are the order in which they were discovered.

Restriction enzymes are powerful tools of moleculargenetics used to:

• Map DNA molecules• Analyze population polymorphisms• Rearrange DNA molecules• Prepare molecular probes• Create mutants

The Type II restriction enzymes recognize specific DNAsequences and cleave the DNA at fixed locations at or nearthe recognition sites. They act as dimers, each subunitrecognizing the same 5' 3' nucleotide sequence incomplementary DNA strands and hence are said to recognizepalindromic sequences. Before the restriction enzyme cuts atits specific recognition site on a long DNA, it binds to a siteamidst a very large number of non-cognate sites. This proteinis then translocated from the initial site to specific site, whichoccurs by "jumping or sliding". At this recognition site in presenceof Mg2+, enzyme undergoes a conformational change, whichkinks the helix and cleaves the DNA producing 'blunt' or 'sticky'ends.

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For e.g. Hae III produced by Haemophilus aegypticuscleaves straight across the double helix when it encounters thefollowing sequence to produce blunt ends.

5'-GGCC-3' 5'-GG CC-3'3'-CCGG-5' 3'-CC GG-5'

However, many restriction enzymes cut in an offset fashionto give sticky ends, which have protruding 5' or 3' ends withunpaired bases depending upon the restriction enzyme, for e.g.EcoR I recognizes the following sequence and cleaves eachbackbone between G and A base residue giving 5' protrudingends.

5'-GAATTC-3' 5'-G AATTC-3'3'-CTTAAG-5' 3'-CTTAA G-5'

Pst I recognizes the following sequence, cleaves between Aand G residue to give 3' protruding ends.

5'-CTGCAG-3' 5'-CTGCA G-3'3'-GACGTC-5' 3'-G ACGTC-5'

The resulting sticky ends can base pair with any DNAmolecule that has the complementary sticky ends to give arecombinant DNA molecule.

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Any restriction endonuclease will cut only a specific basesequence, no matter what DNA molecule it is acting on.However, a given recognition sequence can be recognised bymultiple enzymes called isochizomers. e.g., Sma I andXma I. These can cleave the DNA at same or different positionwithin the recognition sequence. Frequency of cleavagedepends on the probability of occurrence of recognitionsequences. Thus enzyme with longer recognition sequencescut less frequently and consequently produces large fragmentsthan do enzymes with shorter recognition sequences.

Factors affecting Restriction Enzyme Activity:Temperature: Most digestions are carried out at 37°C.However, there are a few exceptions e.g., digestion withSma I is carried out at lower temperatures (~25°C), whilewith Taq I at higher temperature i.e., 65°C.

Buffer Systems: Tris-HCl is the most commonly usedbuffering agent in incubation mixtures, which is temperaturedependent. Most restriction enzymes are active in thepH range 7.0-8.0.

Ionic Conditions: Mg2+ is an absolute requirement for allrestriction endonucleases, but the requirement of other ions(Na+/K+) varies with different enzymes.

Methylation of DNA: Methylation of specific adenine orcytidine residues within the recognition sequence of therestriction enzyme affects the digestion of DNA.

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Kit description:Using this kit, students will perform restriction enzyme

digestion of λ DNA with two different enzymes, EcoR I andHind III. Lambda DNA is a linear double stranded DNA, 48502base pairs, isolated from lambda bacteriophage. It hasrecognition sites for many restriction enzymes and iscommonly used for molecular weight size markers in gelanalysis of DNA and as a substrate in restriction enzymeactivity assays.

Enzymes supplied in this kit, EcoR I and Hind III have5 and 7 recognition sequences on λ DNA. On digestion withEcoR I and Hind III, six and eight fragments of different sizesare obtained respectively. These bands are then resolved byagarose gel electrophoresis to observe the different restrictionenzyme patterns. Simultaneously, a molecular weight markerλ/Mlu I will also be electrophoresed to assess the variousfragment sizes (Refer fig. 1).

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Enzyme Recognition Site on λ Fragment Size (in bp)

EcoR I 21226, 26104, 31749, 39168, 44972

21226, 7421, 5804, 5643, 4878, 3530

Hind III 23130, 25157, 27479, 36895, 37459, 37584, 44141

23130, 9416, 6557, 4361, 2322, 2027, 564, 125



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KT01 : Kit is designed to carry out 5 experiments.Each experiment involves restriction enzymedigestion of λ DNA with EcoR I and Hind III,followed by band pattern analysis by agarosegel electrophoresis. The kit also includeselectrophoresis equipment (ETS-1) requiredfor agarose gel electrophoresis.

KT01A : Kit is designed to carry out 5 experiments.Each experiment involves restriction enzymedigestion of λ DNA with EcoR I and Hind IIIand band pattern analysis by agarose gelelectrophoresis.

KT01B : Kit is designed to carry out 20 experiments.Each experiment involves restriction enzymedigestion of λ DNA with EcoR I and Hind IIIand band pattern analysis by agarose gelelectrophoresis.

Note : Electrophoresis equipment is required forKT01A/KT01B

Duration of experiment: Approximately 4 hours.

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Materials Provided:The list below provides information about the materials

supplied in the kit. The products should be stored as suggested.Use the kit within 6 months of arrival.

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Note: EcoR I & Hind III are supplied as single aliquot

Materials Required:Equipment : Dry bath, Gel rocker (optional).Glassware : Beakers, Conical flask, Measuring cylinder,

Staining tray.Reagent : Distilled water.Other Requirements: Crushed ice/Genei Cooler,

Tips, Micropipette.

Quantity

Materials KT01/01A KT01B Store(5 expts.) (20 expts.)

2X Assay buffer 0.25 ml 1.0 ml -20°C

λ/Mlu I digest 50 µl 0.2 ml -20°C

2.5X Gel loading buffer 0.25 ml 0.25 ml -20°C

Lambda DNA 0.2 ml 0.8 ml -20°C(substrate)

Restriction Enzymes: 15 µl 60 µl -20°C

EcoR I, Hind III

Control λ DNA 0.05 ml 0.2 ml -20°C

Agarose 2.5 g 10 g RT

6X Staining dye 40 ml 160 ml RT

50X TAE 20 ml 80 ml RT

1.5 ml vials 10 Nos. 50 Nos. RT



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Note:• Read the entire procedure before starting the

experiment.• Enzymes are temperature sensitive; hence place the

vials containing enzyme on ice.• Ensure thorough mixing by gently tapping the vial, after

addition of buffer and substrate to the enzyme vial.• Set the dry bath at 37°C prior to starting the experiment.• For preparation of gel, staining and destaining, refer

agarose gel electrophoresis.• Add 5 µl of GLB to 50 µl of λ/Mlu I and 20 µl of GLB to

200 µl of λ/Mlu I digest

Procedure:Setting up the Restriction Digestion Reaction

1. Place the vials containing restriction enzyme (EcoR Iand Hind III) on ice.

2. Thaw the vials containing substrate (Lambda DNA) andassay buffer.

3. Prepare 2 different reaction mixtures using the followingconstituents.

Reaction 1 (EcoR I digestion)• λ DNA 20 µl• 2X Assay Buffer 25 µl• EcoR I 3 µl

Reaction 2 (Hind III digestion)• λ DNA 20 µl• 2X Assay Buffer 25 µl• Hind III 3 µl

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4. Incubate the vial at 37°C for 1 hour.5. Meanwhile, prepare a 1% agarose gel for

electrophoresis.6. After an hour add 5 µl of gel loading buffer to vials.7. Load the digested samples, 10 µl of Control DNA and

10 µl of marker, note down the order of loading.8. Electrophorese the samples at 50-100 V for 1-2 hours.9. Stain the agarose gel with 1X staining dye.

10. Destain to visualize the DNA bands.



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Observation:Observe the band patterns obtained on digestion with

EcoR I and Hind III. Compare these with molecular weightmarker (λ/Mlu I). (Refer fig. 1).

Interpretation:Restriction patterns obtained on digestion with EcoR I

and Hind III are markedly different, demonstrating the factthat each restriction enzyme recognizes and cuts only aparticular base sequence unique to it.

By comparing the migration distances with that of themarker, one can also determine the approximate sizes of DNAfragments.

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1 2 3 4

26,2829,824

5,090

2,2052,419

1,268956

Lane 1 : λ/Hind III DigestLane 2 : λ/EcoR I DigestLane 3 : Control DNALane 4 : λ/Mlu I Digest

Fig 1 : λ DNA digested with EcoR I & Hind III, run on1% Agarose gel (stained with EtBr)

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Agarose Gel Electrophoresis



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Introduction:Agarose gel electrophoresis is a procedure used to

separate DNA fragments based on their molecular weight andis an intrinsic part of almost all routine experiments carried outin molecular biology.

The technique consists of 3 basic steps:• Preparation of agarose gel• Electrophoresis of the DNA fragments• Visualization of DNA fragments

Principle:Preparation of Agarose Gel:

Agarose is a linear polymer extracted from seaweeds.Its basic structure is shown in the figure .

HO CH2O

HO OH

Fig: Basic unit structure of agarose.

Purified agarose is a powder insoluble in water or buffer atroom temperature but dissolves on boiling. Molten solution isthen poured into a mould and allowed to solidify. As it cools,agarose undergoes polymerization i.e., sugar polymerscross-link with each other and cause the solution to gel, thedensity or pore size of which is determined by concentration ofagarose.



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Electrophoresis of DNA fragments:Electrophoresis is a technique used to separate charged

molecules. DNA is negatively charged at neutral pH and whenelectric field is applied across the gel, DNA migrates towardsthe anode. Migration of DNA through the gel is dependent upon:

1. Molecular size of DNA2. Agarose concentration3. Conformation of DNA4. Applied current

Matrix of agarose gel acts as a molecular sieve throughwhich DNA fragments move on application of electric current.Higher concentration of agarose gives firmer gels, i.e., spacesbetween cross-linked molecules is less and hence smaller DNAfragments easily crawl through these spaces. As the length ofthe DNA increases, it becomes harder for the DNA to passthrough the spaces, while lower concentration of agarose helpsin movements of larger DNA fragments as the spaces betweenthe cross-linked molecules is more. The progress of gelelectrophoresis is monitored by observing the migration of avisible dye (tracking dye) through the gel. Two commonly useddyes are Xylene cyanol and Bromophenol blue that migrate atthe same speed as double stranded DNA of size 5000 bp and300 bp respectively. These tracking dyes are negativelycharged, low molecular weight compounds that are loadedalong with each sample at the start of run, when the trackingdye reaches towards the anode, run is terminated.

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Visualization of DNA fragments:Since DNA is not naturally coloured, it will not be visible on

the gel. Hence the gel, after electrophoresis, is stained with adye specific to the DNA. Discrete bands are observed whenthere is enough DNA material present to bind the dye to makeit visible, otherwise the band is not detected. The gel is observedagainst a light background wherein DNA appears as darkcoloured bands.

Alternatively, an intercalating dye like Ethidium bromide isadded to agarose gel and location of bands determined byexamining the gel under UV light, wherein DNA fluoresces.

Note: Ethidium bromide must be handled carefully asit is a mutagen and a carcinogen. Wear gloves whilehandling EtBr solution & gels stained with EtBr.



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Procedure:Preparation of 1% Agarose Gel

1. Prepare 1X TAE by diluting appropriate amount of 50XTAE buffer. (For one experiment, approximately 200 mlof 1X TAE is required. Make up 4 ml of 50X TAE to200 ml with distilled water).

2. Weigh 0.5 g of agarose and add to 50 ml of 1X TAE.This gives 1% agarose gel.

3. Boil till agarose dissolves completely and a clear solutionresults.

4. Meanwhile place the combs of electrophoresis set suchthat it is approximately 2 cm away from the cathode.

5. Pour the agarose solution in the central part of tank whenthe temperature reaches approximately 60°C. Do notgenerate air bubbles. The thickness of the gel should bearound 0.5 to 0.9 cm. Keep the gel undisturbed at roomtemperature for the agarose to solidify.

6. Pour 1X TAE buffer into the gel tank till the buffer levelstands at 0.5 to 0.8 cm above the gel surface.

7. Gently lift the combs, ensuring that wells remain intact.

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Electrophoresis8. Connect the power cord to the electrophoretic power

supply according to the conventionred: anode, black: cathode.

9. Load the samples in the wells in the desired order.10. Set the voltage to 50 V and switch on the power supply.11. Switch off the power when the tracking dye (bromophenol

blue) from the well reaches ¾th of the gel. This takesapproximately one hour.

Staining Procedure to Visualize DNA12. Prepare 1X staining dye by diluting 6X dye (1:6) with

distilled water. (Approximately 50 ml of 1X staining dyeis required for one experiment. Therefore, make up 8 mlof 6X dye to 48 ml with distilled water).

13. Carefully transfer the gel (from gel tank) into a traycontaining 1X staining solution. Make sure that the gel iscompletely immersed.

14. For uniform staining, place the tray on a rocker forapproximately one hour or shake intermittently every10 to 15 minutes.

15. Pour out the staining dye into a container. (The dye canbe reused twice). Destain the gel by washing with tapwater several times till the DNA is visible as a dark bandagainst a light blue background.

Note: Alternatively, Ethidium bromide can be used forvisualizing DNA fragments. Add Ethidium bromide to moltenagarose to a final concentration of 0.5 µg/ml (from a stockof 10 mg/ml in water), when temperature is around 50°C. Mixand cast the gel. After electrophoresis, DNA samples can bevisualized under UV light, they appear fluorescent. Nodestaining is required in this case.



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Ordering Information

Product Size Cat #

GeNeiTM Restriction Digestion 1 Pack KT01Teaching KitConsumables for 5 experiments& Elpho Kit (ETS 1))

GeNeiTM Restriction Digestion 1 Pack KT01ATeaching KitConsumables 5 experiments)

GeNeiTM Restriction Digestion 1 Pack KT01BTeaching KitConsumables 20 experiments)

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