Newcastle University Wetlab/1 September 2008

Monday 1st September

 * To obtain a large enough volume for our ligations, we carried out all 4 restrictions again (i.e. of our pUC57-ncl08, pGFP-rrnB and pJWV021). We used a total volume of 100μl per reaction (using 2μl of each enzyme and 20μl of plasmid).  These were incubated for 1 1/2 hours at 37˚C.


 * 4μl of each reaction was then run on a gel for 1 hour @ 70V- See the gel below (Tuesday the 2nd)

Lane 1: 1kb ladder

Lane 2: pUC57-ncl08 restricted with EcoR1 and Nhe1 (1 hour 30 mins)

Lane 3: pUC57-ncl08 restricted with BglII and Nhe1(1 hour 30 mins)

Lane 4: pGFP-rrnB restricted with EcoR1 and Nhe1(1 hour 30 mins)

Lane 5: pJWV021 restriced with BglII and NHe1(1 hour 30 mins)

Lane 6: 1kb ladder

Lane 7: pUC57-ncl08 restricted with EcoR1 and Nhe1 (2 hours 30 mins)

Lane 8: pUC57-ncl08 restricted with BglII and Nhe1(2 hours 30 mins) Lane 9: pGFP-rrnB restricted with EcoR1 and Nhe1(2 hours 30 mins)

Lane 10: pJWV021 restriced with BglII and NHe1(2 hours 30 mins)

Expected band sizes: * Lane 2: ~ 2.2kb and 2.7kb * Lane 3: ~ 2.2kb and 2.7kb * Lane 4: ~ 7.9kb and 0.5kb * Lane 5: ~ 6.6kb and 0.5kb


 * The gel shows partial restrictions in lanes 2 and 3. In Lane 4, the larger fragment is the correct size, but the observed smaller fragment is larger than expected. Lane 5 shows that no pJWV021 is present, so we will need to re-isolate this tomorrow.


 * The samples were incubated for a further 1 hour, and run on a gel again. Unfortunately, there was no change, so we decided to re-isolate all 3 plasmids tomorrow.


 * We made ON cultures using 10μl ampicillin in 10ml of LB for pUC57-ncl08 and pJWV021, and 5μl of spectinomycin in 10ml LB for pGFP-rrnB. These were incubated overnight at 37˚C.