Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tests for PompC using restriction digest and PCR amplification wit specific primers. Verification of these samples yielded a positive result for PompC-GFP in mini-preped samples.

Our generated fusions proteins lack a Ribosome binding site (RBS) and a promoter, therefore they needed to be added in via overlapping PCR with a Plac template and specific primers. This PCR was attempted in two different manners; one was done in a temperature gradient PCR from 58 degrees to 68 degrees Celsius, and the other was conducted using trouble shooting techniques (such as DNA dilutions and taq polymerase). Both were unsuccessful and no product was obtained.

Continuing amplification of interface components yielded successful amounts of RhlI in several experiments. However, LuxR and LuxI were not amplifying out under normal conditions; therefore several troubleshooting parameters were attempted. Out of four parameters altered, only the increased Magnesium content yielded results. The bands obtained of each product were weak at best. More troubleshooting needs to be attempted. The previously generated fusions (DOM,CON and LIT) were purified, via ethanol purification, as well as Plac.

Troubleshooting PCR amplification of LIT fusion and ETR1 using a temperature gradient (from 58 degrees C to 62 degrees C). Normal PCR amplification of PLac and PompC-GFP. All products are at the correct size!

The Phire DNA polymerase sample we used was obtained from the Dr.Martin lab. Since it was only a trial sample, more Phire DNA polymerase needed to be ordered. We absolutely wanted to order Phire DNA polymerase since it had proven to be successful previously (Aug 01). But when we attempted to order it we figured out that the original Phire, and its second generation 'Phire Hot start Green polymerase' had been discontinued.

We needed ETR1 and EnvZ for Nested Deletion and an amplification of LIT fusion, so an amplification PCR was conducted. For our Nested Deletions; EnvZ was successfully Amplified from E.coli genomic DNA, but ETR1 was not amplified from A. thaliana cDNA. Sadly, our LIT fusion amplification, using DNA template from the successful overlapping PCR, was not successful.

Overlapping PCR; Fusion Generation, from E.coli EnvZ and A. thaliana ETR1, had been giving us problems so we applied a troubleshooting technique using various DNA polymerases. Out of the four polymerases used, only one gave us our Fusions (DOM,CON,LIT) at the expected sizes. 'Phire DNA polymerase' is amazing!