The PCR of the new GUS gene was performed (8 x 20 ul reactions of same proportions as first time round).
A 1% agarose gel was made.
Fig 1. Gel showing another GUS PCR, with a 1Kb+ base ladder in lane 2 and a band of GUS product at roughly 1.8Kb in lane 3.
Took photographs of all Bioassay plates where actinomycete colonies appeared to have a zone of clearing around them - indicating the production of an anti-fungal.
Spore stocks were filtered, concentrated and resuspended in 20 % glycerol.
Isolated Streptomyces sp. colonies from the latest soil sample plates and streaked onto new plates.
Performed some streak purifications and lawns.
Made 2 x 500 ml SFM (+ Ny).
An agarose gel electrophoresis of the PCR products showed a fragment at approx. 1.8 Kb which is likely to be the GUS gene fig 1.
A PCR purification was performed - the product was spun down with PB and PE buffers. The nanodrop indicated that tbere was sufficient DNA to work with.
A restriction digest was performed on pMS82 and the new GUS gene using NdeI and HindIII enzymes in Buffer B. For the GUS gene digest - 5 ul Buffer, 0.5 ul E1 (enzyme 1), 0.5 ul E2, 35 ul template and 9 ul sterile water was used. For the pMS82 digest - 5 ul Buffer, 0.5 ul E1, 0.5 ul E2, 15 ul template and 29 ul sterile water was used.
The plates that had been transformed with the ligation (J04450+neo) contained no colonies.