Newcastle University Wetlab/2 September 2008

Tuesday 2nd September

 * The 3 ON cultures were isolated by dividing each 20ml tube into 2 x 5ml tubes. These were centrifuged for 10 minutes to pellet.


 * Once isolated, we ran 5μl of each sample on a gel to check for the presence of plasmid. As you can see from lanes 1-7 on the gel below, there was plenty of pGFP-rrnB but not much of pJWV021.  Also, the pUC57-ncl08 samples look 'dirty' (see grey smears).


 * As a result, for the restrictions we decided to use more of the pUC57-ncl08 and pJWV021 (in a total volume of 100µl for the restrictions, we used 40µl of plasmid for the pUC57-ncl08 restriction, 30µl for the pJWV021 restriction and 20µl for the pGFP-rrnB restriction).


 * After incubation, we ran the restriction products on a gel.

Expected band sizes: * Lane 9: ~ 7.9kb and 0.5kb * Lane 10: ~ 6.6kb and 0.5kb * Lane 11: ~ 2.2kb and 2.7kb * lane 12: ~ 2.2kb and 2.7kb



Lane 1: 1kb ladder

Lane 2: Unrestricted pGFP-rrnB sample 1

Lane 3: ‘’            ‘’                  sample 2

Lane 4: Unrestricted pJWV021 sample 1

Lane 5: ‘’         ‘’                   sample 2

Lane 6: Unrestricted pUC57-ncl08 sample 1

Lane 7: ‘’               ‘’                   sample 2

Lane 8: 1kb ladder

Lane 9: pGFP-rrnB restricted with EcoR1 and Nhe1

Lane 10: pJWV021 restricted with BglII and Nhe1

Lane 11: pUC57-ncl08 restricted with EcoR1 and Nhe1

Lane 12: pUC57-ncl08 restricted with BglII and Nhe1

The gel showed that our restrictions were successful. Before we moved on to ligating, we purified the 4 restricted samples using the PCR DNA purification kit to remove the enzymes.

For the ligations we made each reaction mixture to 25µl (using 1.5µl of T4 ligase and 2.5µl of 10x ligase buffer). We incubated our samples for 1 hour in a 27˚C water bath.