Week of 4/29-5/3

 Introduction

    Having successfully carried 3 way Gibson Assembly the prior week, the goal of this week was to further this effort by the use of Q5 Polymerase. Q5 Polymerase was used in hopes to amplify results. 

Methods

    PCR using Q5 Polymerase was run on both new Gibson PCR product as well as the prior week's Gibson Assembly. In addition to running PCR on these two products, long amp PCR was also run on PRAD 1 Plasmids in an effort to create more sample for eventual implementation in Gibson with the other three fragments. After having conducted PCR, a gel with 15 wells was run. There were a total of nine samples run (four Gibson PCR products, four long amp PCR products, and a 1kb ladder).

Results

    In the gel pictured below, the well layout is as follows: Well 1- 1kb ladder, Well 2, 3- PCR Gibson Products, Well 4, 5- Original Gibson Assembly Product, Well 6-9- Long Amp PCR.


Conclusions

    Contrary to what was expected, the already PCR'd Gibson Assembly which was run through Q5 Polymerase PCR did not produce any bands. The original Gibson Assembly only put through Q5 Polymerase PCR however did produce brighter bands. On the Long Amp PCR fragments, there were unexpected extra band formations. Further purification of these samples will be conducted in an effort to bring about more desired results.



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