Team:Newcastle/Labwork/6 October 2009

=Formal Lab Session - 6th October 2009= Today we did B. subtilis transformations using our ptotocol. We prepared the LB+ CHL(Chloramphenicol) plates since we will transform the cells with pGFP-rrnB. We used 5ml 2.5 mg/ml stock of CHL, however it should have been 2 ml since the final concentration should have been 5ug/ml CHL.

We did miniprep to look for CotC+ pmutin4 colonies. We used 12 ON cultures. Finally we did a restriction digest with HindIII and BamHI. But we could not find a right colony again. Although the transformations seemed worked we had colonies on both "backbone+ no ligase + no insert" and "backbone+ ligase + no insert". Our transformations dd not have the ligated product! Digest: H20 11ul Buffer 2ul DNa 5ul HindIII 1ul BamHI 1ul

Control Digest: H20 13ul Buffer 2ul uncut pmutin4 2ul HindIII 1ul BamHI 1ul

The bands were pmutin4 were not very clear and we decided to do a midiprep for pmutin4 again.

B.subtilis transformation
1. kinA+pGFP-rrnB from geneArt          10ul    LB+CHL 100ul 2. pGFP-rrnB plasmid                     5ul 3. pSB1AT3                               5ul    LB+CHL 200ul 4. water                                10ul

The concentration of CHL was too high for transformed cells to grow.

E.coli transformation
1. kinA + pGFP-rrnB backbone            20ul    LB+CHL plate 2. psc fragment + pMutin4 backbone      20ul    LB+Amp plate 3. kinA+pGFP-rrnB from geneArt           2ul    LB+Amp plate and LB+CHL plate 4. cotC+pSB1AT3 from geneArt             2ul    LB+Amp plate 5. pSB1AT3                              10ul    LB+Amp plate 6. water                                10ul    LB+Amp plate