My current project is to see if High throughput screening can be used to detect the binding activity of certain genes in the fungal genome. The process involves using PCR to express and amplify the Cpa1 gene with GFP tag. Then to PCR clone the gene, which will then be placed in an expression vector (Drosophila in this case). A SDS-PAGE gel and Western Blot will then be used to confirm that the protein is indeed being produced by the Drosophila system. Large scale protein expression and then the HTP screening will then be conducted.
So far I have been preparing numerous PCR reactions to amplify a gene called cyclophillin A (cpa1) and to soon express this gene with a GFP tag. For the PCRs I have been using Ex Tag enzyme to catalyze the reaction and typically use 0.3 microliters per 50 microliters of reaction mixture. To make sure my PCR products are the right size, I usually run the products on 0.7 to 1.5 % TBE agarose gels. I have also been taught the process of purifying PCR products using the Bench Protocol: QIAquick PCR Purification kit.
I will be continuing this work for the remainder of the semester and into the summer, and hopefully will get some positive results J.