Team:Groningen/Template/MODULE/Notebook/toolbox/week6

The PCR on the BioBrick of the combined genes NisR and NisK was repeated because the yield of this product was very low after the PCR in the week of 4 - 10 August. The mixture of the previous PCR reaction was used as the template. The PCR was performed under standard conditions, with an annealing temperature of 64 °C and 1 μl, 2μl and 3 μl template. No product was obtained with this PCR. So the PCR was once again repeated, this time using diluted template. The mixture was diluted 50x, 300x, 1500x and 15000x. The PCR was repeated using 1 μl and 2 μl of each of the dilutions. This way, product was obtained for all reactions.

The PCR products of the genes NisA, NisB, NisC, NisRK, PNisI and sfGFP(Bs) were purified using the GeneJET purification kit. Then 1 μg of each purified product was restricted with EcoRI and PstI, together with 1 μg of the pSB1C3 plasmid. The restriction enzymes were then inactivated by heating the samples at 80 °C for 20 minutes. Ligated 6 μl of the restricted PCR products to 2 μl restricted pSB1C3. Inactivated the ligase by incubating at 65 °C for 10 minutes. Mixed 5 μl of the ligation mixture with 25 μl electrocompetent Escherichia coli DH5α. The transformants were grown on LB with 10 μg/ml chloramphenicol, see figure 4. The white colonies were tested on insertsize with a colony PCR with the VF2 and VR primers. For every gene transformants were found that contained pSB1C3 with a correctly sized insert, except for NisB and NisRK.

The E. coli that contained the pSB1C3 plasmid with a correctly sized insert were grown as a liquid culture and the plasmid was isolated using the GeneJET Plasmid Miniprep Kit.