PEG PRECIPITATION OF PCR PRODUCTS

PEGPRECIPITATIONOFPCRPRODUCTS ThisprotocolcanbeusedinsteadofEXO/SAPforremovingexcessprimersandnucleotidesfromPCRproductsbeforecycle-sequencing. Protocol: 1.AfterPCR,addthefollowingreagentstoeachtube: For30µlreactionadd: 4.8µlof5MNaCl4.8µlofTEbuffer8.4µlof40%PEG-8000,10mMMgCl2 For50µlreactionadd: 8µlof5MNaCl8µlofTEbuffer14µlof40%PEG-8000,10mMMgCl2 2..AfteraddingthePEG,stirpipetintubeandpipetupanddowntomix.Makesureallsolutionremaininginthetipisinjectedintothetube. 3.Incubateatroomtemperaturefor15minutes. Note: PipetPEGsolutionslowly.PEGsolutionisviscousandmayholdupintip.Ifdoingreactionsinbulk,thethreereagentsmaybemixedandthenaliquotedineither18µlor30µlamountspertube. 4.Pellettheamplificationreactionproductsbycentrifugationat3250rpmfor30minutesat4oC. 5.Removemostofthesupernatantbyinvertingthetrayandcarefullyandgentlydecantingthesupernatant.Removeremainingsupernatantbyinvertingthetray,withpapertowelunderneath,andcentrifugingat300rpmfor3minutes.Repeatwithcleanpapertowel. 6.AirdrythetubesforafewminutesandthenresUSPendeachsamplein25µlofwater.Vortextomix.Load2µlona0.7%agarosegeltoverifythatDNAispresent.Runat55Voltsfor30minutes. 7.Use1µlfordye-terminatorcycle-sequencingreactions.Ifreactionsaretop-heavy,diluteby1/2andrepeatsequencingreaction.