Imperial College/25 September 2008

=25 September 2008=

Cloning

 * EpsE, 5' and 3' AmyE integration sequences digested with EcoRI and PstI




 * All the inserts for EpsE (above) and 5' AmyE (below) appear correct. One of the inserts for 3' AmyE is a different size to the others. Tomorrow 5' and 3' AmyE minipreps will be cut XbaI - SpeI to check for correct orientation. That gel will be run longer with RFP-Terminator (the possible contaminant) to determine if that may be the cause of the size change


 * EpsE Single Colony PCR reactions were cut with HindIII to check that the insert was EpsE and not the possible contaminant, RFP-Terminator




 * The insert in minipreps one and two (numbered left to right) is has lost a small fragment (~150bp) when cut, as should be the case with EpsE which possess a HindIII site about 100-150 away from the 3' end of the gene. The third miniprep shows what appears to be two fragments (one approximately 250bp, the other approximately 400bp), though it is more likely that there are three, as would be the case if RFP-Terminator was the insert instead.