Difference between revisions of "Team:Munich/recbiobricks.html"
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| − | <h4>Assembling | + | <h4>Assembling Biobrick for RecB, C, D</h4> |
<em>2018/08/31 – 2018/09/07</em> | <em>2018/08/31 – 2018/09/07</em> | ||
<table class="table table-borderless"> | <table class="table table-borderless"> | ||
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| − | <h4>Removing PstI | + | <h4>Removing PstI Restriction Site from pSB1C3_RecB</h4> |
<em>2018/09/17-2018/10/02</em> | <em>2018/09/17-2018/10/02</em> | ||
<table class="table table-borderless"> | <table class="table table-borderless"> | ||
Latest revision as of 00:39, 18 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. |