Difference between revisions of "Part:BBa K2446040:Design"

This part was a fusion protein which assembled by three fragments OE-PCR. It contain three core domains from N-terminal to C-terminal: ZF_42-10 DNA binding domain (DBD), nuclear location sequence and transcription regulating domain.

OE-PCR: PCR amplify and connecting the necessary fragments, using proofreading polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temperature. Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.

1.Use proofreading enzyme for extension.

2.Run 10-15 PCR cycles without end primers. (Template extension step)

3.Add end primers, then continue cycling for another 15-20 rounds.

4.Gel extract the correct fragment. Clone into a vector.