BACDNAhasbeenpurifiedsuccessfullyformicroinjectionbythreeprinciplemethods:AnionExchangeChromatography(NucleobondColumns),CsClGrADIent,orSepharose4B-CLChromatography.AnyofthesemethodscanbeusedsuccessfullytopurifyBACDNAforpronuclearmicroinjectionwhentheprotocolsarefollowedcarefully.

Buffercompositionis10mMTris-HCl,pH7.5,0.1mMEDTA,30microMspermine,70microMspermidine,100mMNaCl.Thisbufferismorelikelytoproducetransgenicmicewithintact,unfragmentedDNAmolecules.Seethefollowingreferences:SchedlA,LarinZ,MontoliuL,ThiesE,KelseyG,LehrachH,SchutzG.1993.AmethodforthegenerationofYACtransgenicmicebypronuclearmicroinjection.NucleicAcidsRes21:4783-4787andMontoliuL,BockCT,SchutzG,ZentgrafH.1995.VisualizationoflargeDNAmoleculesbyelectronmicroscopywithpolyamines:applicationtotheanalysisofyeastendogenousandartificialchromosomes..JMolBiol246(4):486-492.SeealsoLluisMontoliu"swebsite.

1000xPolyamineStock

30mMSpermine(Sigma,tetrahydrochloride,#S-1141)70mMSpermidine(Sigma,trihydrocholoride,#S-2501)

Dissolvethespermineandspermidinetogetherinautoclaveddistilledwater,filtersterilize(0.2micronfilters),andstoreat-20C.Sincethepolyaminesareveryhygroscopic,itissuggestedthatsmallquantities(1gram)shouldbeorderedandthenallofitshouldbepreparedatonce.

MicroinjectionBuffer

For50ml:10mMTris-HCl,pH7.50.5mlof1MTris-HCl,pH7.5(autoclaved)0.1mMEDTA,pH8.010microlitersof0.5MEDTA,pH8.0(autoclaved)100mMNaCl1mlof5MNaCl(autoclaved)1xPolyamines50microliters1000xPolyaminesmixAutoclavedH2Oupto50ml

NOTE:Preparefreshanddiscardunusedmicroinjectionbuffer.

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