1 The quantification of GST CaMPARI protein expression via ImageJ in 3 different levels of Arabinose using Restriction mapping Clonase reaction transformation SDS Page and Western blot testing Purpose Starting off with three plates of cell colonies one for each ligation the total number of colonies were counted Then the experimental ligation ligation 1 LG1 was taken and used to perform a culture inoculation Five colonies were taken from LG1 as well as a confirmed positive host to be used as reference and after the inoculation a mini prep was done to all 6 colonies to isolate and purify the plasmid DNA After the purification the clones underwent restriction mapping After the mapping confirmed the presence of desired products the undigested pENTR4 CaMPARI underwent a clonase reaction transformation to generate the bacterial expression vector containing CaMPARI This allowed for bacterial clones that were capable of CaMPARI expression After the clonase reaction 3 cultures were started from the clones in the presences on 02 arabinose 2 arabinose and no arabinose These samples were then run through an SDS page This divided the samples by molecular weight From here a poncceau red stain was done to ensure an even transfer of total proteins on the western blot which was created via an iBlot machine The western blot was then quantified to measure the levels of CaMPARI expression in each of the three levels of arabinose Methods Five bacterial colonies were taken from LG1 and placed into nutrient tubes along with a confirmed positive colony to act as a reference for our 5 test colonies Once the 6 cultures were established a mini prep was performed After the 1 5mL pellet of culture was formed it went through treatment with P2 and N3 Then after flow through was discarded PE buffer was added and then discarded Resulting in uncut pBIT clones 1 6 The plasmid DNA was then quantified by a NanoDrop test as outlined on page 18 of the lab manual Carson et all 2012 From here a RE Digest was performed by placing 10 uL of plasmid DNA in solution with 10 uL of mastermix This mastermix contained restriction endonuclease EcoRV which cut the plasmid DNA at two separate locations The mastermix itself was made up of a solution of dH2O EcoRV and 10x 3 1 EB This cut yielded two blunt ends which allows for forced cloning The solution was then incubated at 37 C for an hour In order to deactivate the enzyme loading dye was added to the solution From here a gel electrophoresis test was performed following the procedure found on pages 27 28 and 31 of the lab manual Carson et All 2012 After the gel test confirmed the content of both the vector and insert and the NanoDrop test measured concentration and purity clone 4 was selected to continue the experiment as it had a high concentration and was the most pure according the the A260 A280 ratio of 1 84 A solution made of clone 4 underwent a clonase reaction pDEST15 and 2 uL of clonase were added to the reaction This sample was vortexed centrifuged and then incubated at room temperature for an hour After an hour proteinase K was added to solution to inhibit the clonase from further reacting After the clonase reaction was completed the plasmid DNA was transformed into competent cells 50 uL of E coli BL21 AI was added to the clonase mixture After incubation the mixture was then heat shocked to allow for absorption of the plasmid DNA into the competent cells After a second brief ice incubation 80 uL of LB was added to solution 2 From here the samples were shaken for 1 hour at 37 C Finally 100 uL of cells were plated onto selective LB media LB amp Arabinose The cells were plated according to the process outlined in page 39 of the lab manual Carson et all 2012 After cell colonies were successfully expressed on the plates cultures with 2 arabinose 02 arabinose and no arabinose were created from successful colonies that were previously plated These 3 samples were treated with culture and 2X sample buffer and mixed by pipetting After the solution was thoroughly mixed it was incubated at 95 C for ten minutes Afterwards 50 uL of supernatant was collected To test the purity of our proteins an SDS Page test was prepared Each of 3 the three samples were loaded into two separate lanes to improve testing results The gel was created by following the procedure outlined in page 93 of the lab manual Carson et all 2012 The samples and molecular weight standards were prepared for the test as outlined in pages 93 and 94 of the lab manual Carson et all 2012 The Gel was then ran at 60mA 30mA per gel as two gels are run simultaneously for an SDS Page test After the test was complete the gels were taken to the Trans illuminator for UV activation so that the protein bands could be thoroughly viewed In order to transfer the proteins from the acrylamide gel an Invitrogen iBlot was employed to transfer proteins via dry transformation onto a nitrocellulose membrane This was accomplished by following the outline as depicted in a worksheet provided in the lab Following the iBlot transfer a poncceau red stain was performed following the method outlined in page 100 of the lab manual Carson et all 2012 Once a successful protein transfer had been completed to the PVDF membrane the membrane underwent blocking and incubation The membrane was soaked with ethanol washed with TBS T and then incubated for 15 minutes on an orbital shaker Afterwards the blocking solution was removed from the membrane From here the membrane was immediately incubated with the primary antibody rabbit anti GST Ab at a 1 20000 dilution and then again with the secondary antibody anti rabbit mixed with HRP and 2 uL of Ab at a 1 5000 dilution After each incubation with antibodies the membrane was washed repeatedly with TBS T The treatment with antibodies followed the outline as provided in pages 100 and 101 of the lab manual Carson et all 2012 After the completion of both incubations the membrane was intended to undergo Luminescence detection However before the membrane could be treated with ECL it was discovered that either the 1 Ab or the 2 Ab was faulty and thus the Luminescence detection would not be successful In order to test what substance was at fault the membrane was exposed to the primary antibody at a more extreme solution This second exposure was done at a 1 1000 The gel was then left overnight to incubate in lab On return to the lab it was determined that it was neither the primary nor secondary Ab s fault for the inability