In Search of My Father - edvotek.com · In order to match the boys with the correct parents, chromosomal DNA fingerprinting tests were ordered for the boys, the mothers, and the surviving

S-49.151009

S-49Edvo-Kit #S-49

In Search of My FatherExperiment Objective:

Students will learn how agarose gel electrophoresis separates different sizes of dye molecules that represent DNA fragments. They will learn how these fragments form unique DNA patterns for each person, which is the basis for solving maternity and paternity identity.

See page 3 for storage instructions.

Page

Experiment Components 3

Experiment Requirements 3

Background Information 4

Experiment Procedures Experiment Overview 6 Agarose Gel Electrophoresis 8 Critical Thinking and Hypothesis Development & Study Questions 10 Instructor's Guidelines Overview of Instructor's Pre-Lab Preparations 11 Pre-Lab Preparations 12 Experiment Results and Analysis 13 Study Questions and Answers 14

Appendices 15 A EDVOTEK® Troubleshooting Guide 16 B Bulk Preparation of Agarose Gels 17 C Practice Gel Loading 18

Safety Data Sheets can be found on our website: www.edvotek.com/Safety-Data-Sheets

Table of Contents

In Search of My Father EDVO-Kit #S-49

1.800.EDVOTEK • Fax 202.370.1501 • [email protected] • www.edvotek.com

2

Duplication of any part of this document is permitted for non-profit educational purposes only. Copyright © 1989-2015 EDVOTEK, Inc., all rights reserved. S-49.151009


Experiment Components

EDVOTEK and The Biotechnology Education Company are registered trademarks of EDVOTEK, Inc. Ready-to-Load, QuickStrips and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.

READY-TO-LOAD™ SAMPLES FOR ELECTROPHORESISStore QuickStrip™ samples in the refrigerator immediately upon receipt.

All other components can be stored at room temperature.

Components (in QuickStrip™ format) Check (√)

A Standard dyes with assigned base pair equivalents q B Mother 1 DNA q C Mother 2 DNA q D Boy 1 DNA q E Boy 2 DNA q F Father (surviving, married to Mother 1) q REAGENTS & SUPPLIES

• PracticeGelLoadingSolution q• UltraSpec-Agarose™ q• ElectrophoresisBuffer q• 1 ml pipet q• 100 ml graduated cylinder (packaging for samples) q• Microtipped Transfer Pipets q

Experiment #S-49 is designed for 10 gels.

Store QuickStrip™ samples in the refrigerator immedi-ately upon receipt. All other components can be stored at room temperature.

• Horizontalgelelectrophoresisapparatus• D.C.powersupply• Automaticmicropipetswithtips(optional)• Balance• Microwave,hotplateorburner• Pipetpump• 250mlflasksorbeakers• Hotgloves• Visualizationsystem(whitelightbox)• Distilledordeionizedwater

All experiment components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor admin-istered to or consumed by humans or animals.

Requirements

In Search of My FatherEDVO-Kit #S-49

3




Background Information

In a war torn country, two young boys were separated from their respective parents who had been imprisonedbytheregime.Thechildrenwerecousinswhowereborntwomonthsapartandlookedstrikinglysimilar.Theirmotherswerehalfsisterswho sharedacommonmother(Figure1).After several years, the regime was overthrown and replaced by a new government that released allpoliticalprisonersfromprison.Theprisonerswere reinstated in their respective communities andallchargesagainstthemweredropped.

During this period, the two boys had not been separated from each other, and had been adopted by a high-ranking military officer who hadnochildrenofhisown.Boththeofficerandhis wife died in a liberation uprising and for a short period of time the boys were placed in an orphanage.Ontheir18thbirthday,theboyswerereleasedfromtheorphanageandtheyimmediatelybegantosearchforinformationregardingtheirlostparents.

Thebrotherscontactedanumberofeldersfromtheirvillage.Theeldersinformedthemthataftertheirbiologicalparents were released from prison, one of the fathers was assassinated by individuals who were sympathetic to thefallenregime.Thesecondbiologicalfatherwasfoundinthevillage,sufferingfromamnesia.Twowomen,who were thought to be their biological mothers, were in a rehabilitation facility that housed approximately 200 women.Uponarrivaltothecenteritwasveryobviousthatthepatientshadnotreceivedpropermedicalattention.Basedonageandappearance,abouttenwomenfittheprofileofthetwomothers.

DETERMINATION OF PARENTAGE USING DNA FINGERPRINTING

First, the boys sought to determine which of the ten women were their mothers by performing mitochondrial DNAfingerprinting.Mitochondria,the“powerhouse”ofthecell,areuniqueorganellesinthattheycontainasmallDNAgenome.Thisgenomeisusefulforidentifyingmaternitybecausemitochondriaareinheritedthroughthefemaleline.Beforeconception,ahumaneggcontainsalargenumberofmitochondria.Incontrast,humanspermcontainsveryfewmitochondria.Uponfertilizationofthehumaneggbyasperm,thedevelopingzygotecontainsmitochondriaobtainedfromthemother’segg.

Mitochondrial DNA fingerprinting tests can be used as an initial screening technique because they are less expensivethanchromosomalDNAtestingandresultsareavailableinashorterperiodoftime.Inthiscase,sincethe boys were cousins (their mothers were half-sisters who shared the same mother), the mitochondrial testing resultswouldidenticalforthetwoboys.ThetestsidentifiedtwowomenwithmitochondrialDNAfingerprintingpatternsthatmatchedthatoftheboys.

Grandma's First

Husband

Grandma

Mother 1

Child

Mito

chon

drial D

NA M

atch

Grandma's Second Husband

Child

Mitochondrial DNA M

atch

Mother 2

Father 1

Boy Cousins

Father 2

Figure 1: Pedigree of the Boys in Question



4



In order to match the boys with the correct parents, chromosomal DNA fingerprinting tests were ordered for theboys,themothers,andthesurvivingfather.ChromosomalDNA,whichispresentinthenucleusofeverylivingcell,isthegeneticmaterialthatactsasablueprintforalloftheproteinssynthesizedbythatcell.UnlikemitochondrialDNA,chromosomalDNAisanequalcompositeofbothparents.Ineachchromosomepair,oneisinheritedfromthefatherandthesecondfromthemother.AlthoughmostofthisDNAisidenticalbetweenindividuals,smallsequencedifferences,or“polymorphisms”,occuratspecificlocationsthroughoutthegenome.ThesepolymorphismsincludesinglebasepairchangesandrepetitiveDNAelements.Byexaminingseveralofthesepolymorphicregions,wecangenerateaunique“DNAfingerprint”forthatperson.

DNAfingerprintscanallowustodistinguishoneindividualfromanother.Becausepolymorphismsareinherited, DNA fingerprints can also be used to determine paternity/maternity (and other familial relation-ships).Todetermineparentage,theDNAfingerprintsoftheboysarecomparedwiththeDNAfingerprintsfromthesurvivingfatherandthetwomothers.SincechromosomalDNAisinheritedfrombothparents,theDNAfingerprintofachildwillcontainamixtureofpolymorphismsfromeachparent.ThepatterniseasilyrecognizableasmatchingvisibleDNAbandsonthegelandcanbeexperimentallydemonstratedinthissimulationexperiment.

Background Information

5




EXPERIMENT OBJECTIVE:

StudentswilllearnhowagarosegelelectrophoresisseparatesdifferentsizesofdyemoleculesthatrepresentDNAfragments.TheywilllearnhowthesefragmentsformuniqueDNApatternsforeachperson,whichisthebasisforsolvingmaternityandpaternityidentity.

WORKING HYPOTHESIS

If DNA samples collected from different mothers and fathers are examined at variable polymorphic sites, then one should be able to match the children with their real mother andfatherbytheDNAfingerprintingmethod.

LABORATORY SAFETY

1. Glovesandgogglesshouldbewornroutinelyasgoodlaboratorypractice.2. Exerciseextremecautionwhenworkingwithequipmentthatisusedinconjunctionwiththeheatingand/or

meltingofreagents.3. DONOTMOUTHPIPETREAGENTS-USEPIPETPUMPS. 4. Exercisecautionwhenusinganyelectricalequipmentinthelaboratory.5. Alwayswashhandsthoroughlywithsoapandwaterafterhandlingreagentsorbiologicalmaterialsinthe

laboratory.

LABORATORY NOTEBOOKS:

Scientists document everything that happens during an experiment, including experimental conditions, thoughts andobservationswhileconductingtheexperiment,and,ofcourse,anydatacollected.Today,you’llbedocument-ingyourexperimentinalaboratorynotebookoronaseparateworksheet.

Before starting the Experiment: • Carefullyreadtheintroductionandtheprotocol.Usethisinformationtoformahypothesisforthis experiment. • Predicttheresultsofyourexperiment.

During the Experiment: • Recordyourobservations.

After the Experiment: • Interprettheresults–doesyourdatasupportorcontradictyourhypothesis? • Ifyourepeatedthisexperiment,whatwouldyouchange?Reviseyourhypothesistoreflectthischange.

Experiment Overview

Wear gloves and safety goggles



6



Experiment Overview

7




1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A).2. MIX agarose powder with 1X buffer in a 250 ml flask (see Table A).3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like water).4. COOL agarose to 60° C with careful swirling to promote even dissipation of heat.5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well template (comb) in the appropriate notch.6. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. The gel will stiffen and become less transparent as it solidifies.7. REMOVE end caps and comb. Take particular care when removing the comb to prevent damage to the wells.

60°C

1:001. 3.

4. 5.

7.

Caution! Flask will be HOT!

Concentratedbuffer

Distilledwater

Agarose

2.50x

Flask

© 2013 Edvotek® All Rights Reserved.

ConcentratedBuffer (50x)

Size of GelCasting tray

7 x 7 cm

7 x 10 cm

7 x 14 cm

0.6 ml

1.0 ml

1.2 ml

+DistilledWater

29.4 ml

49.0 ml

58.8 ml

+TOTALVolume

30 ml

50 ml

60 ml

=

Individual 0.8% UltraSpec-Agarose™ GelTable

A

60°C20min.

WAIT6.

Pour

Amt ofAgarose

0.23 g

0.39 g

0.46 g

Agarose Gel Electrophoresis

1. DILUTEconcentrated(50X)bufferwithdistilledwatertocreate1Xbuffer(seeTableA).2. MIXagarosepowderwith1Xbufferina250mlflask(seeTableA).3. DISSOLVEagarosepowderbyboilingthesolution.MICROWAVEthesolutiononhighfor1minute.Care-

fully REMOVEtheflaskfromthemicrowaveandMIXbyswirlingtheflask.ContinuetoHEAT the solution in 15-secondburstsuntiltheagaroseiscompletelydissolved(thesolutionshouldbeclearlikewater).

4. COOLagaroseto60°Cwithcarefulswirlingtopromoteevendissipationofheat.5. Whileagaroseiscooling,SEALtheendsofthegel-castingtraywiththerubberendcaps.PLACE the well

template(comb)intheappropriatenotch.6. POUR the cooled agarose solution into the prepared

gel-castingtray.Thegelshouldthoroughlysolidifywithin20minutes.Thegelwillstiffenandbecomelesstransparentasitsolidifies.

7. REMOVEendcapsandcomb.Takeparticularcarewhen removing the comb to prevent damage to the wells.

IMPORTANT:

If you are unfamiliar with agarose gel prep and electrophoresis, detailed instructions and helpful resources are available at www.edvotek.com

ConcentratedBuffer (50x)

Size of GelCasting tray

7 x 7 cm

7 x 10 cm

0.6ml

1.2 ml

+DistilledWater

29.4 ml

58.8 ml

+TOTALVolume

30 ml

50 ml

=

Individual 0.8% UltraSpec-Agarose™ Gel

Amt ofAgarose

0.24 g

0.48 g

Table

A




8



Agarose Gel Electrophoresis

8. PLACEgel(onthetray)intoelectrophoresischamber.COVER the gel with 1X electrophoresis buffer (See Table B for recommended volumes).Thegelshouldbecompletelysubmerged.

9. PUNCTUREthefoiloverlayoftheQuickStrip™withapipettip.LOAD the entire sample (35-38 µL) into the well in consecutive order.TheidentityofeachsampleisprovidedinTable1.

10.PLACEsafetycover.CHECKthatthegelisproperlyoriented.Re-member, the negatively charged dye samples will migrate toward thepositive(red)electrode.

11.CONNECT leads to the power source and PERFORM electrophoresis (SeeTableCfortimeandvoltageguidelines).

12. Afterelectrophoresisiscomplete,REMOVE the gel and casting tray from the electrophoresis chamber and VISUALIZE theresults.Nostainingisnecessary.

1X DilutedBuffer

8. 9.

10. 11. 12.( - )

( + )

1 2 3 4 5 6

Pour

Lane

1

2

3

4

5

6

Tube A

Tube B

Tube C

Tube D

Tube E

Tube F

Table 1: Gel Loading

Standard Dye Markers

Mother 1

Mother 2

Boy 1

Boy 2

Father

50x Conc.Buffer

DistilledWater+

EDVOTEKModel #

Total Volume Required

1x Electrophoresis Buffer (Chamber Buffer)

M6+

M12

M36 (blue)

300 ml

400 ml

500 ml

Dilution

Table

B

6 ml

8 ml

10 ml

294 ml

392 ml

490 ml

M36 (clear) 1000 ml 20 ml 980 ml

Reminders:

If unfamiliar with gel loading, consider performing the optional activity in Appendix C, Practice Gel Loading, prior to performing the experiment.

Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.

Staining is not required for Experiment #S-49, but results must be analyzed upon completion of the electrophoretic separation. Because dye molecules are extremely small they will diffuse out of the gel. Thus, the gel cannot be saved.

Time and Voltage Guidelines(0.8% Agarose Gel)

Recommended TimeVolts

1257050

20 min.45 min. 90 min.

Table

CElectrophoresis of Dyes

9




Critical Thinking and Hypothesis Development

1. Whatisthevariableinthisexperiment?

2. Whatisthecontrolinthisexperiment?

3. Whatcouldonechangeintheexperimentifthisexperimentwasrepeated?

4. Writeahypothesisthatwouldreflectachange.

5. Basedontheevidenceobtainedfromtheanalysisofthegel,whichchild,motherandfatherarereunited?Explain.

Study Questions

1. Whatdothedifferentdyebandsthatwereseparatedbyelectrophoresisrepresent?

2. Whydodifferentindividuals,suchassiblings,havedifferentfingerprints?

3. WhatisthebasisofmitochondrialDNAfingerprintanalysis?

4. WhatisthedifferencebetweenmitochondrialandcellDNA-basedfingerprintinganalysis.


10



Instructor's Guide

11



INSTRUCTOR'S GUIDEEDVO-Kit #S-49 In Search of My Father

OVERVIEW OF INSTRUCTOR’S PRELAB PREPARATION:

This section outlines the recommended prelab preparations and approximate time requirement to complete each prelabactivity.

What to do: When: Time Required:

Prepare diluted Electrophoresis Buffer

Prepare molten agarose and pour gel

40 min.

Prepare QuickStrips™

Up to one day before performingthe experiment.

Pre-Lab Preparations:

Each Student Groupshould receive:• Electrophoresis Buffer (50x)• Distilled Water • UltraSpec-Agarose™• Ready-to-Load™ Samples

NOTE:Accurate pipetting is critical for maximizing successful experi-ment results.

If students are unfamiliar with using micropipets, we recom-mend performing the optional activity found in Appendix C, Practice Gel Loading, prior to conducting the experiment.

Carefully cut betweeneach set of tubes

EDV

OTE

K®

•

DO

NO

T BE

ND

A

B

C

D

E

F

G

H

CU

T H

ERE

A

B

C

D

E

F

G

H

CU

T H

ERE

A

B

C

D

E

F

G

H

CU

T H

ERE

CU

T H

ERE

A

B

C

D

E

F

G

H

CU

T H

ERE

A

B

C

D

E

F

G

H

A

B

C

D

E

F

G

H

SEPARATION OF PCR PRODUCTS BY AGAROSE GEL ELECTROPHORESIS

Thisexperimentrequiresa0.8%agarosegelperstudentgroup.Youcanchoosewhethertopreparethegelsinadvanceorhavethestudentspreparetheirown.Allowapproximately30-40minutesforthisprocedure.

Individual Gel Preparation:

Each student group can be responsible for casting their own individual gel prior toconductingtheexperiment.SeetheStudent’sExperimentalProcedure.Students will need Electrophoresis Buffer (50x) concentrated buffer, distilled waterandagarosepowder.

Batch Gel Preparation:

To save time, a larger quantity of agarose solution can be prepared for sharing bytheclass.Electrophoresisbuffercanalsobepreparedinbulk.SeeAppendixB.

Preparing Gels in Advance:

Gelsmaybepreparedaheadandstoredforlateruse.Solidifiedgelscanbestoredunderbufferintherefrigeratorforupto2weeks.

Donotfreezegelsat-20ºCasfreezingwilldestroythegels.

Gelsthathavebeenremovedfromtheirtraysforstorageshouldbe“anchored”back to the tray with a few drops of molten agarose before being placed into the tray.Thiswillpreventthegelsfromslidingaroundinthetraysandthechambers.

SAMPLES FORMAT: PREPARING THE QUICKSTRIPS™

QuickStrip™tubesconsistofamicrotiterblockcoveredwithaprotectiveover-lay.Eachwellcontainspre-aliquoteddyes.

Usingsharpscissors,carefullydividetheblockoftubesintoindividualstripsbycuttingbetweentherows(seediagramatright).Takecarenottodamagetheprotectiveoverlaywhileseparatingthesamples.

Eachlabgroupwillreceiveonesetoftubes.Beforeloadingthegel,remindstudentstotapthetubestocollectthesampleatthebottomofthetube.

FECBA D


12


INSTRUCTOR'S GUIDE In Search of My Father EDVO-Kit #S-49

Experiment Results and Analysis

13



INSTRUCTOR'S GUIDEEDVO-Kit #S-49 In Search of My Father

S-49 gel result photo

3,500

1,500800

450

1 2 3 4 5 6

B1

Y1

B2

Y1

Y2RP

B3

Y2 R R

Y1P P

ThepurplebandofBoy2matchesthepurplebandofMother1.TheredbandofBoy2matchestheredbandofthefather.Therefore,Boy2isthechildofMother1andthefatherinthisscenario.

TheloweryellowbandfromBoy1matchestheloweryellowbandfromMother2.NeitherofthebandsfromBoy1matchthefather.Therefore,Boy1isthechildofMom2andthedeceasedfather.

Color legend

B1 Blue 1B2 Blue 2B3 Blue 3P Purple 1R RedY1 Yellow1Y2 Yellow2

In the two idealized schematics, the relative positions of dye molecules are shown but are not depicted to scale.

3,500

1,500800

450

1 rehtoM

2 rehtoM

1 yoB2 yoB aF

rehtkra

M

sre

Lane Tube Sample

1

2

3

4

5

6

Tube A

Tube B

Tube C

Tube D

Tube E

Tube F

Standard Dye Markers

Mother 1

Mother 2

Boy 1

Boy 2

Father

Please refer to the kit insert for the Answers to

Study Questions

A EDVOTEK® Troubleshooting Guide

B Bulk Preparation of Agarose Gels

C Practice Gel Loading

Safety Data Sheets can be found on our website: www.edvotek.com/Safety-Data-Sheets

Appendices

15



APPENDICESEDVO-Kit #S-49 In Search of My Father

Appendix AEDVOTEK® Troubleshooting Guides

PROBLEM: CAUSE: ANSWER:

Bands not visible on the gel

Ensure that the electrophoresis buffer was correctly diluted.

The electrophoresis buffer was not prepared properly.

Dye bands disappearwhen the gels are keptat 4° C.

Gel was not prepared properly. Make sure to prepare a 0.8% gel.

The dye molecules are small and will diffuse out of the gel.

The results must be analyzed upon the completion of electrophoresis

Very light colored bandseen after electrophoresis

Pipetting error.Make sure students pipet 35 µl of dye sample per well.

Poor separation of bands

Ensure that leads are attached in the correct orientation.

The dyes ran off of the gel because the polarity of the leads was reversed.

Contact the manufacturer of the electrophoresis unit or power source.

Malfunctioning electrophoresis unit or power source.


16


APPENDICES In Search of My Father EDVO-Kit #S-49

Appendix B

To save time, the electrophoresis buffer and agarose gel solution can be prepared in larger quantities for sharing by the class.Unuseddilutedbuffercanbeusedatalatertimeandsolidifiedagarosegelsolutioncanberemelted.

Bulk Preparation of Agarose Gels

Bulk Electrophoresis Buffer

Quantity (bulk) preparation for 3 liters of 1x electro-phoresisbufferisoutlinedinTableD.

Batch Agarose Gels (0.8%)

Forquantity(batch)preparationof0.8%agarosegels,seeTableE.

1. Usea500mlflasktopreparethedilutedgelbuffer

2. Pour3.0gramsofUltraSpec-Agarose™intothepreparedbuffer.Swirltodisperseclumps.

3. Withamarkingpen,indicatethelevelofsolutionvolumeontheoutsideoftheflask.

4. Heattheagarosesolutionasoutlinedpreviouslyforindividualgelpreparation.Theheatingtimewillrequireadjustmentduetothelargertotalvolumeofgelbuffersolution.

5. Cooltheagarosesolutionto60°Cwithswirlingtopromoteevendissipationofheat.Ifevaporationhasoccurred,adddistilledwatertobringthesolu-tionuptotheoriginalvolumeasmarkedontheflaskinstep3.

6. Dispensetherequiredvolumeofcooledagarosesolutionforcastingeachgel.ThevolumerequiredisdependentuponthesizeofthegelbedandDNAstainingmethodwhichwillbeused.RefertoAppendixAorBforguidelines.

7. Allowthegeltocompletelysolidify.Itwillbecome firm and cool to the touch after approxi-mately20minutes.Thenproceedwithpreparingthegelforelectrophoresis.

60˚C

Note: The UltraSpec-Agarose™ kit component is usually labeled with the amount it contains. Please read the label care-fully. If the amount of aga-rose is not specified or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount.

50x Conc.Buffer +

DistilledWater

Total Volume Required

60 ml 2,940 ml 3000 ml (3 L)

Bulk Preparation of Electrophoresis BufferTable

D

Batch Prep of 0.8% UltraSpec-Agarose™Table

EAmt ofAgarose

(g)

ConcentratedBuffer (50X)

(ml)+

DistilledWater(ml)

TotalVolume

(ml)+

3.0 7.5 382.5 390

17




Appendix CPractice Gel Loading

Accuratesampledeliverytechniqueensuresthebestpossiblegelresults.Pipettingmistakescancausethesampletobecomedilutedwithbuffer,orcausedamagetothewellswiththepipettipwhileloadingthegel.

If you are unfamiliar with loading samples in agarose gels, it is recommended that you practice sample delivery tech-niquesbeforeconductingtheactualexperiment.EDVOTEKelectrophoresisexperimentscontainatubeofpracticegelloadingsolutionforthispurpose.Castingofaseparatepracticegelishighlyrecommended.Onesuggestedactivityisoutlined below:

1. Castagelwiththemaximumnumberofwellspossible.

2. Afterthegelsolidifies,placeitunderbufferinanelectrophoresisapparatuscham-ber.

Alternatively, your teacher may have cut the gel in sections between the rows of

wells.Placeagelsectionwithwellsintoasmall,shallowtrayandsubmergeitunderbufferorwater.

3. Practicedeliveringthepracticegelloadingsolutiontothesamplewells.Takecarenottodamageorpuncturethewellswiththepipettip.

• Forelectrophoresisofdyes,loadthesamplewellwith35-38microlitersofsample.

• Ifusingtransferpipetsforsampledelivery,loadeachsamplewelluntilitisfull.

4. Ifyouneedmorepractice,removethepracticegelloadingsolutionbysquirtingbufferintothewellswithatransferpipet.

5. Replacethepracticegelwithafreshgelfortheactualexperiment.

Note: If practicing gel loading in the electrophoresis chamber, the practice gel loading solution will become diluted in the buffer in the apparatus. It will not interfere with the experiment, so it is not necessary to prepare fresh buf-fer.

Note: The agarose gel is some-times called a "submarine gel" because it is submerged under buffer for sample loading and electrophoretic separation.


18




2-20 µl

20-200 µl

100-1000 µl

2 0.0

2 – 20 µl

tens, ones, tenths (in decimal)

2 0.0

20 – 200 µl

2 0 0hundreds, tens, ones

100 – 1000 µl

1 0 0 0thousands, hundreds, tens, ones

2 0 0

1 0 0 0

© All rights reserved, Edvotek, Inc. 2013


SETTING THE VOLUME OF AN ADJUSTABLE VOLUME MICROPIPET

1. CHOOSEthecorrectmicropipetforthevolumeyouaremeasuring.MakesurethatthevolumetobemeasuredDOES NOT EXCEEDtheupperorlowervolumesettingofthemicropipet.

2. DETERMINEtheunitsmeasuredbythemicropipetbylookingatthevolumesetting.Thesettingwillappearinthewindowonthesideofthemicropipet.Notethatthedifferentmicropipetsusedifferentscalesfortheirmeasure-ments.Somemicropipetsareaccuratetoatenthofamicroliter,whileothersareaccuratetoonemicroliter.

3. SETthevolumebytwistingthetopoftheplunger.Ingeneral,twistingtheplungerclockwisereducesthevolume,andtwistingtheplungercounterclockwiseincreasesthevolume.

19






MEASURING LIQUIDS WITH A MICROPIPET

1. SETthemicropipettotheappropriatevolumebyadjustingthedial.

2. PLACEacleantiponthemicropipet.

3. PRESStheplungerdowntothefirststop.HOLD the plunger down while placing the tip beneaththesurfaceoftheliquid.

4. SlowlyRELEASEtheplungertodrawsampleintothepipettetip.Positionthepipettipoverthewell.Becarefulnottopunctureordamagethewellwiththepipettip.

5. DELIVERthesamplebyslowlypressingtheplungertothefirststop.Depresstheplungertothesecondstoptoexpelanyremainingsample.DO NOT RELEASE the plunger until the tipisoutofthebuffer.

6. DISCARD thetipbypressingtheejectorbutton.Useanewcleantipforthenextsample.

4 5 6Dial

1 2 3

2 0.0

2 0

.0

2 0

.0

2 0

.0

2 0

.0

2 0

.0

2 0

.0


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Documents

In Search of My Father - edvotek.com · In order to match the boys with the correct parents, chromosomal DNA fingerprinting tests were ordered for the boys, the mothers, and the surviving