TemplatePreparation

Thequalityofsequencingresultsisdirectlyrelatedtothequalityofthetemplate.ABIrecommendsaminialkaline-lysis/PEGprecipitationprocedure(theCorecansupplyinformationonthisprotocol).ABIalsohasaplasmidpreparationkitwhichtheyrecommend.Severalothercompaniesmarketsimilarkits.Mostofthesewillworkforautomatedsequencing.Youshouldexperimentandusethemethodthatworksbestinyourlab.

TheCorecanalsoprovideinformationonpurifyingPCRproductsforsequencing.

NOTE:EDTAisapotentinhibitorofthesequencingreaction.Templateinbufferwith1mMEDTAisacceptable.Thefinalconcentrationinthesequencingreaction(20ul)willthenbe0.5mMorlessdependingonthevolumeoftemplateadded.AfinalEDTAof1mMorhigherwillcompletelyinhibitthereaction.

PossIBLeTemplateProblems

Templatecontaminationbycellularconstituentsduetopoortechnique.Templatedegradation-nucleasecontamination.Improperlyquantitatedtemplate-everytemplateshouldbequantitatedbyanOD260andvisualizedonagel.Excesssaltinthetemplatefrompurificationprotocol-40mMsaltseverlyinhibitsthesequencingreaction.EDTAinthetemplate-1mMEDTAcompletelyinhibitsthesequencingreactionbychelatingcriticalcations.Multipletemplatesinthepreparation-actuallyoccursfrequentlyincloningexperimentswhereprecautionsforclonalidentityarenottaken;resultsinmultiplepeakspernucleotideposition.

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