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Describe the principles of In-frame cloning

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Biology

Describe the principles of In-frame cloning

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Answer The gene of interest usually has to be amplified from genomic orvector DNA by PCR polymerase chain reaction before it can becloned into an expression vector The first step is the design ofthe necessary primersImportant features arePrimer sequence Especially the 3end of theprimer molecule is critical for the specificity and sensitivity ofPCR It is recommended not to have3 of more G or C bases atthis position This may stabilize nonspecific annealing of theprimera 3 thymidine since it is more prone tomispriming than the other nucleotidesPrimer pairs should be checked for complementarity at the3end This often leads to primerdimer formationBases at the 5end of the primer are less critical for primerannealing Therefore it is possible to add sequenceelements like restriction sites to the 5end of theprimer moleculePrimer length Usually a primer length of1830 bases is    See Answer
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