APh$162$–$Molecular$Biology!Day$2$!Restriction!Digests!The$LacZ$gene$o n$our$plasmid$is$flanked$by$restriction$sites$for$the$enzyme$`KpnI’$on$one$side$and$`HindIII’$on$ the$other,$allowing$us$to$cut$the$gene$out$and$leave$precisely$con trolled$‘sticky‐en ds’$on$the$plasmid$DNA,$onto$which$we$ will$ligate$our$new$reporter$gene$(or$`insert’),$called$$%Venus(YFP).$$The$enzyme$KpnI$cuts$at$any$site$ with$the$sequence$$...5‐GGTAC’C‐ 3...$$leaving$the$sticky‐end$GTAC,$while$the$enzyme$Hi ndIII$cuts$at$any$site$with$ the$sequ ence$...5‐A ’AGCTT‐3...$leaving$the$sticky‐end$AGCT.$$$The$fact$that$these$sticky$ends$are$distinct$means$we$can$dis cour age$the$plasmid$from$ligating$on$itself,$and$ensure$that$we$i nsert$the$new$reporter$gene$in $the$correct$orientation.$$$A$my riad$of$available$rest riction$enzymes$e nsur es$that$we$ca n$control$each$piece$o f$the $plasmid$independen tly.$$ In$today’ s$lab$session, $we$will$digest$pZE21‐lacZ$and$the$YFP $(Venus)$PCR$amplicon$from$day$1.$$Restriction$ enzyme$concentrations$are$me asured$in$the$units$of$`activity’,$wh ere$1$uni t$of$enzyme$can$digest$1$µg$of$DNA$in$1$hour$at$37°C.$$To$ensure$that$all$of$the$DNA$is$cut $by$t he$enzyme,$it $is$generally$recommended$to$add$enzyme$in $excess$o f$the $required$amount.$$Follow$the$protocol$below,$and$mix$the$fo llowing$reagents$to$begin$this$enzymatic$reaction.$$Once$the$reaction$is$fi nished,$we$w ill$examine$the$products$o n$an$agarose$gel$t o$determine$their $size$and$differences$in$topology.$$To$p roperly$gauge$the$size$of$DNA$segments$that$appear$on$t he$agarose$gel,$we $need$to$add$a$so‐called$`DNA$Ladder’$that$contains$segments$of$known$length,$against$which$we$will$compare$our$plasmid$digest.$ $We$will$use$ two$com mercially$available$ladder s,$an d$we$wil l$cre ate$our$own$`DNA$Ladder’$using$the$distinct$enzyme$EcoRI$to$digest$$λ–phage$(viral)$genomic$DN A$that$has$be en$pre‐digested$with$HindIII.$$$$Today$you$will$perform$the$following$rest riction$digests:$1. KpnI/HindIII$double$digest$of$the$PCR‐generated$YFP$( Venus)$insert$from$Da y$1.$2. KpnI$single$dige st$of$vector$pZE21‐LacZ$3. HindIII$single$digest $of$vector$pZE21‐LacZ$4. KpnI/HindIII$do uble$digest$of$vec tor$pZE2 1‐lacZ$5. EcoRI$single$dig est$of$pre‐HindIII$digested$lambda$ phage$DNA$$/Double/ Dige st/of/YFP/(Venus)/inser t/$Reagent:$Amount:$YFP$DNA$(from$Day$1$PCR)$200$ng$NEB$Buffer$2$(10X)(NEBEB7002S)$1X$BSA$(10X)$$$1X$KpnI$(10$units/µl)$$2$µl$HindIII$(10$units/µl)$$2$µl$DDH2O$Calc.$Total$Volume$50$µl$//Double/ Dige st/of/pZE21%lacZ/vector/%/$Reagent:$Do uble$Digest:$Kpn$I$S ingle$Digest$Hind$III$Single$Digest$No $Digest$Control$Plasmi d$(pZE21_LacZ$)$DNA*$300$ng$300$ng$300$ng$300$ng$NEB$Buffer$2$(10X)$$1X$1X$1X$1X$BSA$(10X)$1X$1X$1X$1X$KpnI$(10$units/ul)$$1$µl$1$µl$0$µl$0$µl$HindIII$(10$units/ul)$$1$µl$0$µl$1$µl$0$µl$DDH2O$Calc.$Calc.$Calc.$Calc.$Total$Volume$30µl$30µl$30µl$30µl$*$Your$TA$will$provide$the$concentration.$$λ/–/phag e/Digest$$Reagent:$Amount:$Digested$λ$DNA$0.5$ug/µl$1.5$µg$NEB$EcoRI$Buffer$(10X)$$$$1X$BSA$(10X)$$$1X$EcoRI$(10$units/µl)$$2$µl$DDH2O$Calc.$Total$Volume$30µl$$No tes:$$$NEB$Buffer$2$is$one$of$many$potential$buffers,$chosen$to$optimiz e$the$performance$of$both$KpnI$an d$HindIII.$$$See$“Enzyme$Properties”$at:$$htt p://www.neb.com/nebecomm/products/productR0104.asp$htt p://www.neb.com/nebecomm/products/productR0142.asp$$Procedure:$1. Verify$all$ca lculated$ reagent$volumes$with$you r$TA.$2. For$the$given$concentration$of$input$DNA,$calc ulate$the$corresponding$volume.$3. Mix$this$volume$of$DNA$along$with$th e$specified$amounts$(see$tables$above)$of$buffer,$res triction$enzymes$and$water$to$brin g$the$total$volume$to$25$µl,$in$ a$small$tube.$$Wh en$finished,$ask$your$TAs$for$the$app ropriate$amounts $of$restriction$enz yme$$$4. Mix$thoroughly$ and$spin$in$the$micro‐centrifuge$for$a$few$seconds$(<6$sec)$to$sediment$all$the$con tents.$5. In$addition$to$using$commercial$ladders,$we$will$create$our$own$ladder$using$lambda$phage$DNA.$a. The$λEphage$genom e$is$48,5 02bp,$if$the$sequence$was$totally$random,$how$many$HindIII$(5A’AGCTT3) $and$Eco RI$(5G’A ATTC3)$sites$would$ you$expect?$$Ho w$many$are$there$in$rea lity?$$i. Given$the$actual$restriction$sites$for$EcoRI$and$HindIII,$ calculate$the$expected$segme nt$lengths$of$ the$DNA$ladder.$ii. See$the$attached$restriction$site$map$for$the$λ‐ phage$ge nom e.$$6. Place$all$six$of$your$tubes$in$a$37°C$incubato r$for$1$hour.$$ During$this$incubation,$we$will$prepare$the$agarose$ gel.$$$DN A!Agarose!Gel!Electrophoresis!The$above$enzymatic$reactions$and$accom panying$controls$have$produced$a$range$of$DNA$ lengths$and$the$no‐enzy me$(uncut$DNA)$control$e ven$has$a$distinct$polymer$topology.$$It$is$immensely$useful$to$be $able$to$measure$the$l engt hs$produ ced$during$DNA$manipulations.$$The$pr imary$method$em ploy ed$throughout$molecular$biology$is$to$subject$DNA$i n$an$ultra‐viscous$agarose$gel$ to$an$ele ctric $field,$wherein$we$utilize$t he$~‐5.88e/nm$charge$on$DNA $to$‘pull’$ it$throug h$the$gel.$$In$a$given$period$of$time$ the$DNA$migrates$through$the$gel$roughly$proportional$to$the$inverse$of$its$ma ss.$$$$$1X$TAE$Buffer$~500$ml$1X$TAEE$1%$agarose$gel$~$40$ml$(40$mL$TAE$+$0.4g$agarose)$100bp$DNA$Ladder$(NEBEN0467S)$2$µl$ladde r$+$3$µl$H2O$+$1$µl$6x$loading$dye$1000bp$DNA$Ladder$(NEBEN3232)$2$µl$ladde r$+$3$µl$H2O$+$1$µl$6x$loading$dye$For$plasmi d$digest,$3$controls,$λ$DNA$25$µl$of$digest$+$5$µl$6x$loading$dye$No te:$$TAE$(Tris‐acetate$EDTA)$buffer$$Materi als:$gel $box$$ gel $power$suppl y$gel $tray$$ gel$comb$$$$/////////////You$will$run$the$following$ sam ples $on$the$gel:$1. 100$bp$ DNA$ladder$2. Non‐purified,$non‐digest ed$PCR$product$from$day$1$3. Lambda$phage$double$digest$4. pZE21‐LacZ$–$KpnI$single$dig est$5. pZE21‐LacZ$–$KpnI$single$dig est$6. pZE21‐LacZ$–$KpnI/HindIII$double$digest$7. pZE21‐LacZ$–$no$enzyme$control$ digest$8. 1$k b$DNA$la dder$b)$a)$DNA$ Ladders$a)$$Example$of $1$kb$DNA$ladder$on$a $0.8%$a garose$gel,$stain ed$with$EtBr.$b)$$Example$of$the$100bp$DNA$ladder$on$a$1.3%$aga rose$gel,$stained$with$Et Br.$$$$$Proced ure:!$1. Using$DI$water,$clean$the$gel$box, $gel$comb$and$gel $tray.$2. In$short$bursts$of$~10s,$heat$ the$1X$TAE‐$1%$agarose$gel$in$the$microwave$until$i t$is$completely$liquefied. $3. Secure$the$gel$comb$in$the$gel$tra y$and$orient$so$th at$the$re