Newcastle University Wetlab/11 August 2008

Monday 11th August

 * Plasmids were isolated from pUc57-ncl08 (the ncl08 fragment contains the spaRK 2-part component system). We aim to clone this into pGFP-rrnB and pJWV021 vectors.


 * Restrictions of pGFP-rrnB and pJWV021 carried out using 10μl plasmid and 0.5μl in 50μl total reaction volume (see Restricting Plasmids (Double Restriction)). pJWV021 was restricted in a 2-step reaction as we beleived that the enzymes were incompatible in the same buffer. The second restriction of this plasmid used 48μl of the purified plasmid mixture in a total volume of 100μl. pGFP-rrnB was restricted using EcoRI and NHeI. pJWV021 was restricted firstly with NHeI and secondly with BglII.

These were purified and stored overnight at -25˚C.