Nested Deletions Using ExonucleaseIII and Mung Bean Nuclease

1. Anestedseriesofdeletionscanbeproducedby:cuttingplasmidDNA(2.5ugpertimepoint,seebelow)withanenzymeleavingablunt(egEcoRV)or5""overhand(egEcoRI)onthesidethatthedeletionsaretoproceed.Topreventdeletionsintheotherdirectioncutwithanenzymeleavinga3""overhang(eg.Kpn.NOTE:thatSacIIwillnotworkforthispurpose)orcuttingwitha5""overhangcreatingenzymeandfillingwiththionucleiotidesasfollows.Thisismorereliablethanusinga3""overhang:
   1. Cutwiththeenzymetobefilledin
   2. Heat70°Cfor15min.Quenchonice.
   3. Addthio-dNTPto40uMand5Uklenow
   4. 20minatRT.Heatto65°Cfor10min.
   5. Phenol/CHCL3extractandethanolppt.
   6. Cutwiththesecondenzymeonthesidethatdeletionsaretoproceedinaround100ul.Heatinactivatetheenzymewhencomplete.Alinearizedcloneandlinearizedvector(1ugea.)provideusefulMarkersforthestartandendofthedeletionseries,respectively,whenthedeletionsarerunonagel.
2. ResUSPendtheDNAin

   -	1Xexobuffer(seeend)
   -	10mM2-mercaptomethanol(preparedfreshfrom14Mstock)
   -	XulDDW
   -	Thevolumeshouldequal25ulpertimepoint.Heatto30°for5mintoequilibrateatincubationtemp.
   -	Dispense20ul10Xmungbeanbuffer,155ulDDWintoEppendorftubesforeachtimepoint.
   -	Haveonhandcrusheddryice.

3. Add20UofExoIIIperugofDNAandvortex.
4. Remove25ulaliquot’sintothedilutedmungbeanbufferatXsecintervalsaccordingtothefollowingtableandplaceondryice.

   -	37°=400bp/min
   -	34°=375bp/min
   -	30°=230bp/min
   -	23°=125bp0/min

   -	Igenerallydomyincubationsat30°Candtakemyfirstaliquotat20secandthereafterat40-80secintervals(atestforthebesttimepointsmaybeuseful).

5. Whenfinishedremovetubesfromdryiceandheatto68°Cfor15minthenplaceonice.
6. TomaketheDNAbluntended,add7.5Umungbeannuclease(dilutedinmungbeandilutionbuffer,seeend)per2.5ugtimepointandincubateat30°for30min.
7. Ethanolppte,resuspendandloadontoa0.8%TAEgel.RunandexcisebandsfreefromundigestedDNA.IsolateDNAandligateovernight.Transformandpick2-4coloniespertimepointforminigelanalysisandsubsequentsequencing.

Buffers

Comments:

1. TheDNAmustbe>85%supercoiled.ExoIIIwilldigestfromnicks.UseCsClpurifiedDNAandavoidusingtheUVlamptoisolateplasmidsfromCsClgrADIents.ChecktheDNAonagelvs.cutplasmid.
2. TheDNAmustbefullyrestrictedwiththe5""overhang(otherwisebackgroundofundigestedDNA)andwiththe3""overhang(otherwisedeletionsproceedsinbothdirections).
3. UseageltoisolatethedeletionfragmentsratherthansimplyextractingtheDNA(Extractionofthedeletionsafterthemungbeansteprequirestheadditionof4ul20%SDS,10ul1MTrispH9.5,20ul8MLiCland250ulphenol/CHC1₃,followedbyre-extractionandprecipitation)afterthemungbeanstep.Gelpurificationispreferableasitremovesthebackgroundandallowsyoutomonitorhowwellthereactionhasproceeded.