This web page was produced by Zack Perfect as an assignment for an undergraduate course at Davidson College

Determining Orientation of Plasmid

            Once PCRed, the gene of interest IDH1, is ligated into the plasmid. Because the sticky edges of the plasmid are the same, being cut with the same restriction enzyme, the gene could insert into the plasmid either right side up (forward) which is desired, or upside down (reverse). In order to determine the orientation we took samples from various colonies of E. coli we grew containing different possible insert orientations. We used restriction enzyme that cut once inside the gene and once outside, therefore producing differing sizes of DNA strands depending on the orientation. We used Sal I; below are predictions of the framents under the two different scenarios. When the digestions are run on gel electrophoresis, two bands should appear and by determining their sizes, the orientation should be confirmed. The samples containing forward orientation can be used to proceed with the experiment.

Digested with Sal I

forwardpic.jpg (14939 bytes)     reversepic.jpg (14850 bytes)


forwardcut.jpg (9826 bytes)     reversecut.jpg (9640 bytes)



Other predicted fragment sizes

IDH2 cut with EcoRV     Forward: ~ 320 bp     Reverse: ~ 790 bp

IDP1 cut with BamHI     Forward: ~ 507 bp      Reverse: ~ 780 bp

IDP2 cut with BglII     Forward: ~ 743 bp      Reverse: ~ 496 bp

IDP3 cut with Cla I then BamHI     Forward: ~ 522 bp      Reverse: ~ 795 bp