Difference between revisions of "Team:Munich/recbiobricks.html"
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<td>Notes:</td> | <td>Notes:</td> | ||
| − | <td> | + | <td>Because of time restraints, this experiment was done using ligation and gibson assembly approaches in parallel <br> |
PCR: RecB_BB_PstI-remove-Lig | PCR: RecB_BB_PstI-remove-Lig | ||
Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv | Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv | ||
Revision as of 16:48, 17 October 2018
Assembling biobrick for RecB, C, D
2018/08/31 – 2018/09/07| Participants: | Enikö Baligács |
| Protocol: | PCR, Agarose gel, Gel extraction, Ligation, Chemical transformation, Mini Prep |
| Notes: | Three PCRs for three BioBricks with primers: RecD_BB_EcoRI_fw & RecD_BB_SpeI_rv (expected: 3,5 kb) RecB_BB_EcoRI_fw & RecB_BB_SpeI_rv (expected: 1,8 kb) RecC_BB_EcoRI_fw & RecC_BB_SpeI_rv (expected: 3,3 kb) AT 68° ET 70 s; restriction digest of pSB1C3_mRFP with EcoRI and SpeI and PCR purification of the Backbone each PCR was digested with EcoRi and SpeI and ligated into the Backbone. |
| Results: | correct bands were cut out. Ligations gave colonies. Test PCR and sequencing confirmed ligation, sent more primers for complete insert sequencing. Completely sequenced positive clones were obtained. |
Removing PstI restriction site from pSB1C3_RecB
2018/09/17-2018/10/02| Participants: | Enikö Baligács |
| Protocol: | PCR, PCR purification, Ligation, Gibson assembly, Transformation |
| Notes: | Because of time restraints, this experiment was done using ligation and gibson assembly approaches in parallel PCR: RecB_BB_PstI-remove-Lig Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv Templ: RecB_BioBrick Plasmid AT: 72° ET: 11 min PCR: RecBCD_BB_PstI_remove_lig Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv Templ: RecBCD_BioBrick Plasmid AT: 72° ET: 11 min PCR: RecBCD-TEV_BB_PstI_remove-Lig Primer: RecB_mut_Lig_fw & RecB_mut_Lig_rv Templ: RecBCD-TEV_BioBrick Plasmid AT: 72° ET: 11 min PCR: RecB_BB_PstI-remove-GA Primer: RecB_mut_fw & RecB_mut_rv Templ: RecB_BioBrick Plasmid AT: 72° ET: 11 min PCR: RecBCD_BB_PstI_remove_GA Primer: RecB_mut_fw & RecB_mut_rv Templ: RecBCD_BioBrick Plasmid AT: 72° ET: 11 min PCR: RecBCD-TEV_BB_PstI_remove-GA Primer: RecB_mut_fw & RecB_mut_rv Templ: RecBCD-TEV_BioBrick Plasmid AT: 72° ET: 11 min |
| Results: | Colonies were obtained, but sequencing showed incorrect cut site assembly. Because of time restrictions these attempts were not continued and we focused on other parts of the project. |