The PETTOPO manual is loaded onto the Bb course content page as is the Alpha- Am

Question

The PETTOPO manual is loaded onto the Bb course content page as is the Alpha- Amylase Genbank sequence. The PETTOPO Manual gives specific directions for primer design for plasmid pET101/D-TOPO as summarized on pages 9-12 and p44. The forward primer must have a sequence that overlaps that of the plasmid (as summarized in the manual) and must contain an ATG start codon. Print your primer in the 5' to 3' direction below and list the location of the corresponding base pairs of the Amylase sequence. The reverse primer must eliminate the stop codon at the end of the Amylase sequence but keep the open reading frame (coding sequence) of the amylase protein in-frame with the coding sequences in the plasmid (the V5 epitope and poly-Histidine regions). Print your primer in the 5' to 3' direction below and list the location of the corresponding base pairs of the Amylase sequence.

Explanation / Answer

Reverese Primer: In bold sequence GTGG is hoverhang and complementary sequence of cloning vector while underline CTA is representing stop codon.

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