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Cycle Sequence Reactions For Large Insert Plasmid Templates

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Thefollowingdye-labeledterminatorreactionchemistrieshavebeendesignedtobalanceconservationofreagentswiththeresultingsequenceproductsignalstrength.WhencoupledwithBACPACResourcetemplatepreparation,N-freereadlengthsof>400ntscanbeexpected.Moreemphasiscanbeplacedonreagentconservationbyfurthervolumereductionordilution;oronsequenceproductsignalstrengthbyincreasingthereactionsize.

CycleSequenceChemistryConditions:

Chemistry³	ReactionSize⁴	FinalVol(ul)	PrimerVol(10pmol/ul)	ReactionMixvol(ul)	Templatevol(ul)
ABIdRhodterm	1.25x	25	1	10	14
ABIBDterm	0.75x	15	1	6	8


CycleSequenceConditions:
1)Initialdenaturationstep:96^oC4min.2)denaturation:96^oC10sec.3)annealing:50^oC10sec.4)extension:60^oC4min.runsteps2-4for100cycles.5)hold:4-10^oC.
PrimersUsefulForBACandPACVectors:
T7.29:5’gccgctAATacgactcactatagggagag29merT7:5’taatacgactcactataggg20mergSP6:5’gttttttgcgatctgccgtttc22mer

³StockABIReadyReactionmixesaresupplementedwith4mMMgCl2⁴ReferstoABI1x(20ul)reaction⁵Annealingtempcanbeadjusted,basedonTmofprimersused

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Cycle&nbsp;Sequence&nbsp;Reactions&nbsp;For&nbsp;Large&nbsp;Insert&nbsp;Plasmid&nbsp;Templates

Cycle Sequence Reactions For Large Insert Plasmid Templates