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Team:Newcastle/Labwork/6 October 2009
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(→Formal Lab Session - 6th October 2009) |
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The bands were pmutin4 were not very clear and we decided to do a midiprep for pmutin4 again. | The bands were pmutin4 were not very clear and we decided to do a midiprep for pmutin4 again. | ||
| + | === Experiment procedure === | ||
| + | ==== 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 | ||
{{:Team:Newcastle/Project/Labwork/CalTemplate}} | {{:Team:Newcastle/Project/Labwork/CalTemplate}} | ||
Revision as of 01:29, 21 October 2009
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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 "backbnone+ no ligase + no insert" and "backbnone+ 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.
Experiment procedure
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
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