TheCorecanalsoprovideinformationonpurifyingPCRproductsforsequencing. NOTE:EDTAisapotentinhibitorofthesequencingreaction.Templateinbufferwith1mMEDTAisacceptable.Thefinalconcentrationinthesequencingreaction(20ul)willthenbe0.5mMorlessdependingonthevolumeoftemplateadded.AfinalEDTAof1mMorhigherwillcompletelyinhibitthereaction. PossIBLeTemplateProblems Templatecontaminationbycellularconstituentsduetopoortechnique.Templatedegradation-nucleasecontamination.Improperlyquantitatedtemplate-everytemplateshouldbequantitatedbyanOD260andvisualizedonagel.Excesssaltinthetemplatefrompurificationprotocol-40mMsaltseverlyinhibitsthesequencingreaction.EDTAinthetemplate-1mMEDTAcompletelyinhibitsthesequencingreactionbychelatingcriticalcations.Multipletemplatesinthepreparation-actuallyoccursfrequentlyincloningexperimentswhereprecautionsforclonalidentityarenottaken;resultsinmultiplepeakspernucleotideposition.TemplatePreparation
Thequalityofsequencingresultsisdirectlyrelatedtothequalityofthetemplate.ABIrecommendsaminialkaline-lysis/PEGprecipitationprocedure(theCorecansupplyinformationonthisprotocol).ABIalsohasaplasmidpreparationkitwhichtheyrecommend.Severalothercompaniesmarketsimilarkits.Mostofthesewillworkforautomatedsequencing.Youshouldexperimentandusethemethodthatworksbestinyourlab.
