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How big is the insert? How many base pairs are there between the two restriction enzyme sites on the pET28a?

mpark

How big is the insert? How many base pairs are there between the two restriction enzyme sites on the pET28a?

If the insaert DNA is generated from PCR, try to make blunt ending then gel purification of the blunt-ended 825 bp PCR product. make blunt end ligation of the 825 bp fragment on some subclone vector, such pUC19 first. Screening subclones and sequencing the positive one. Cut off the insert DNA and then clone it into pET28a.

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mpark

If the insaert DNA is generated from PCR, try to make blunt ending then gel purification of the blunt-ended 825 bp PCR product. make blunt end ligation of the 825 bp fragment on some subclone vector, such pUC19 first. Screening subclones and sequencing the positive one. Cut off the insert DNA and then clone it into pET28a.

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