Bad Primer - (Jun/27/2005 )

I have had a reverse primer made and used it in pcr. When I ordered it it contained the penultimate codon, stop codon, HindIII site, a 3bp stuffer, NotI site and 3bp clamp.

I pcr'd and cloned and sequenced. The T from the stop codon TAG is missing in all my sequences. Everything else is there just as it should be.

Most possible a bad primer or is there another explanation?

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Does the stop codon match to your template?

this could happen



template: AATC-AGGC
primer: AATCTAGGC

product: AATCAGGC


If not, the T may be missing from the primer during synthesis.

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I am not aware that this could happen. Could you explain how a base can be cleaved out a primer during pcr.

It simply looks like a bad primer and needs to be reordered.

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