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!!APh$162$–$Molecular$B iology!!Day$1$$PCR$(Polymerase$Chain$Reaction)$of$the$Insert$!In!this!step! we!will!ampl ify!the!insert!DNA!and!prepare !it!fo r!res triction!digest.!!See!the!include d!‘Phusion!HF!Ma stermix‘!handouts!for!more!details .!!Total!V olume:!!50!ul!Reagent:$Amount:$Insert$Plasmid$(pZS2LacUV501_Venus$ @$25$ng/µl)$DNA$(YFP)*$100$ng$Forward$Primer$(10$uM)$2.5$ul$Reverse$Prime r$(10$uM)$2.5$ul$Phusio n$HF$Mastermix$$(2X)$1X$DDH2O$Calc.$*Replace!the!YFP!D NA!with!an!equivalen t!amount!of!water!in!the!no!t emplate!control!react ion.!$You$will$set$up $three$reactions:$two$identical$reactions$as$described$ab ove$ and$one$“no$template$control.”$$The$no$template$control$reaction$is$the$same$as $the$normal$reaction,$but$leaves$out$the$YFP$template$(sterile$water$is$used$instead).$$Why$do$you$thi nk$this$is$an$important$control$to$perform?$$Procedure:!1. In!thin-walled!PCR!tubes,!mix!the!DNA,! primers,! mastermix!and!water!together.!!Bri efly!centrifug e!thi s!tube!to !pull!all!liquid!to!the!bottom.!!We!will!call!this!tube!the!‘PC R!reaction.’!!Labe l!your!tubes!f or!the!three!react ions!as!clearl y!as!possible!!2. Hopefully!the!id ea!of!thermally!cycling!during!PCR!is!conceptually!clear,!now!we!must!program!the!actual !device!to!perform!this!task!–!your!TA!will!help!you!with!this.!!Step:$Temp$(C):$Time:$1$a$Initial$DNA$Denaturation$98$45$s$2$a$Denaturat ion$98$8$s $3$a$Annea ling$62$10$s$4$a$Ex tension$72$15$s$5$a$Final$Extension$72$5$min$6$a$Hold$$4$Indef.$Steps!2!–!4!will!repe at!fo r!a!total!of!35!times.!!$$Ho w$much$total$amplif ication$i s$that?$$What$limits$ maximum$amplification?$!3. You!will!run!one!of!the!reactions!and!the!no!te mplate!control!reaction!on!an!agarose!gel !during!the!next!day!of!molecular!b iology.!!The!third!reac tion!will!be!purified,!as!described!below.$$PCR$purification$of$the$insert$$The!PCR!reactions!you!perfor med!now!contain!the!amplified!insert!DNA!that !you!want!to!res triction!digest!in!the!next!step!of!the!subcloning—however,!they!also !contain!a!lot !of!other!things!(e.g.,!free!nucleo tides)!that!decrease!the!efficiency!of!restriction!enzy mes.!!Because!of!this,! you!will! need!to!purify!the!amplified!DNA!u sing!a!simple!procedure!and!the!QIAquick!PCR!purificatio n!kit!from!Qiagen.!!You!will!only!be!pur ifying!one!of!the!reactions!(do!not !purify!the!no!templat e!control!).!!1. Add!5!volumes!of!Buffer! PB I! to!1! volu me!of!the!PCR!sample!and!mix.!For2example,!add!500!μl!of!Buffer!PBI!to!100!μl!PCR!sample.!2. To!bind!DNA,!apply!the!s ample!to!the!QIAquick!col umn !and!centrifuge!for!60!s!at !ma ximum!speed.!3. Discard!flow‐through.!Place!the!QIAquick!column!back!into!the!same!tube.!Collection!t ubes!are!re‐used!to!reduce!plastic!wast e.!4. To!wash,!add!0.75!ml!Buffer!PE!to!the!QIAquic k!column!and!cen trifuge!for!60!s!at!ma ximum!speed.!5. Discard!flow‐through!and!place!the!QIAquick!column!b ack!in!the!same!tube.!Centrifuge!t he!empty!col umn !for!an!additional!1!min!at!max imum!speed.!IMPOR TANT:!Residual!ethanol!from!Buffer!PE!will!not!be!completely!removed!unless!the!flow‐ through!is!discarded!befor e!thi s!additional!centrifugation.!6. Place!Q IAquick!column!in!a!clean!1.5!ml!microcentrifuge!tube.!4. To!elute!DNA,!carefully!add!35!μl!Buffer!EB!(10!mM!Tris·Cl, !pH!8.5)!to!th e!center!of!the!QIAquick!membrane,!let!the!column!stand!for!1!min,!and!then!ce ntrifuge!the!col umn !for!1!min!at!maximum!speed.!!IMPOR TANT:!E nsur e!that!the!elution !buffer!is!dispensed!directly!on to!the!QI Aquick!me mbrane!for!complete!elution!of!bound!DNA.!Ask!your!TA!to!check!that!you!have!done!this!properly!before!your!final!spin.!$NanoD rop$$As! a!final!step,!y ou!will!use!the!NanoDrop! spec trophotometer!to!determi ne!the!concentration!of!your!purified,!PC


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