Team:Groningen/Template/MODULE/Notebook/toolbox/week4

The CP promoters in pSB1C3, that were isolated in the week of 14 - 20 July, were tested on their insert size. For this, a PCR was done on the plasmid using the primers VF2 (BBa_G00100) and VR (BBa_G00101) were used. The insert size corresponded to the expected size.

A touchdown PCR was done on the genes of the nisin operon and the sfGFP(Bs) gene. In this PCR the annealing temperature dropped from 55 °C to 45 °C, lowering the temperature with 1 °C each cycle. This unfortunately resulted in a PCR program of just 10 cycles, instead of the intended 30. The remaining steps of the program were finished the next morning. Only the genes NisA, PNisI and sfGFP(Bs) were amplified this way, see figure 1. Therefore, the PCR was repeated, this time with a complete cycle. No additional genes were amplified this way.

Another attempt was made for amplification of the remaining genes. This time, the most ideal annealing temperature for each gene was used by using a gradient PCR and placing the tubes at the optimal temperature. This still did not amplify the remaining genes. Also, a PCR with a general annealing temperature of 50 °C was done. This also did not amplify the remaining genes.

The genes that were amplified in the first PCR (NisA, PNisI and sfGFP(Bs)) were purified using the GeneJET PCR Purification Kit from Thermo Scientific. The purified products were digested with the enzymes EcoRI and PstI, using 2 μl of the product. These purified and restricted products were loaded on gel, see figure 2. It was then discovered that the genes were barely visible on gel. The restricted genes were purified with the GeneJET PCR Purification Kit and the concentration was measured with the NanoDrop 1000. The clean, restricted products were too low in concentration to be suitable for ligation.