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Newcastle University Wetlab/1 September 2008
From 2008.igem.org
Newcastle University
GOLD MEDAL WINNER 2008
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Wet lab work was carried out from 4 August to 19 September, Mondays to Fridays. Please click on a day to see the lab notebook.
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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.
