Trouble Shooting Davidson Design

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Problem with Design:

We designed the half edges with BsaI sites in both the middle and the ends of the half edges. Therefore when we digest with BsaI and perform Golden Gate shuffling we will get complete edges that are incorrect and to long.

See General Primer to design to look at the problem in out design Media:ggsdiagram.pptx‎

Also, could be explained as followed:

We could now get get B-AA-BB-AA-BB-A When we want A-BB-A

Possible Solution:

After using Golden Gate Shuffling to create our complete edges we will perform another round of PCR with original primers. This will create many different sizes of the correct sequence of complete edges. Then we can run a gel and gel purify for the correct complete edge based on the number of bp.

Therefore all edges with A-BB-A in the sequence would be amplified. We could get A-BB-A, A-AA-BB-A, A-AA-BB-AA-BB-A, etc. But would could screen for length and only select A-BB-A

To Avoid this Problem in the Future:

We need to create two sticky ends one with BsaI and another with a different restriction enzyme that serves the same purpose. Therefore, when we digest with BsaI only the part of the half edges we want to ligate together will during Golden Gate Shuffling.

Possible Enzymes Available To Create Sticky Ends Similar to BsaI: Media:AlternativeRestrictionEnzymes.png