Agarose gel electrophoresis showed that something had gone wrong during PCR - genomic DNA was visible.
A PCR reaction was repeated using 8 x 10 ul reactions of the same Master Mix proportions as before. A gradient PCR was performed this time where the annealing temperature was altered for each reaction in order to optimise the PCR.
Made 6 x 500 ml SFM (500 ul Ny per 500 ml). Autoclaved for 1 hr and poured into plates.
Made a 1% agarose gel.
Performed more spore stocks of lawned plates.
Diluted soil sample 85 to a concentration of 10^-4 and plated 100 ul of 10^-2 to 10^-4 dilutions on SFM + Ny agar plates.
As the agarose gel from the 22/08 was unsuccessful, the restriction digest of 3b and AntGpneo with PstI and NdeI was repeated. The miniprep BL21 cells that contain pETAntA and AntGpRFP plasmids were digested with XbaI. All samples were analysed by gel electrophoresis.
The gel was successful and a large fragment from 3b and small fragment from AntGpneo were excised, purified and ligated overnight at 4°C. Colonies one and two from the plate transformed with BL21 (pET28AntA + AntGRFP) cells looked unusual on the gel. They appeared to contain unexpected DNA. But Colonies three and four from the same plate looked promising and would need further investigating to establish whether they contain two plasmids.
Two colonies from the plates containing samples 3a and Bba_J04450 were inoculated into 10ml cultures of LB and Chlorophenicol antibiotic. The four cultures (two colonies from each) were left to grow overnight at 37°C.