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Team:Hannover/Notebook/Plant Vector
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<p class="text">Here we list all the steps that were tested to modify the pORE E3 <a href="http://www.ncbi.nlm.nih.gov/nuccore/AY562536">(AY562536.1)</a> into a vector with BioBrick <a href="http://parts.igem.org/Help:Standards/Assembly/RFC21">RFC[21]MCS</a> and 2x35S promotor.</p> | <p class="text">Here we list all the steps that were tested to modify the pORE E3 <a href="http://www.ncbi.nlm.nih.gov/nuccore/AY562536">(AY562536.1)</a> into a vector with BioBrick <a href="http://parts.igem.org/Help:Standards/Assembly/RFC21">RFC[21]MCS</a> and 2x35S promotor.</p> | ||
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Revision as of 04:56, 14 October 2014
Notebook / Plant Vector
Here we list all the steps that were tested to modify the pORE E3 (AY562536.1) into a vector with BioBrick RFC[21]MCS and 2x35S promotor.
date |
coworkers |
lab |
activity |
short summary |
| 25-Sept-2014 | Lisa | IPG | colony-PCR | colony-PCR with primer 1261 and 488: all 24 colonies negative or inconclusive/ no more time to do the insertion of rcf again |
| 24-Sept-2014 | Steffen, Lisa | IPG | triple digest/ oligo annealing | vector: triple digest: gel extraction/ insert: oligo annealing/ phosphorylation/ ligation of vector and insert/ transformation of XL1-Blue Competent Cells/ incubation over night (25 °C)/ everything performed after modified protocol |
| 23-Sept-2014 | Steffen | IPG | plasmidpreparation/ amplification of pORE_E3 | plasmidpreparation of ONC from Katharina/ amplification |
| 22-Sept-2014 | Lisa, Katharina | IPG | insertion of RFC[21] | colony-PCR: only negative controls/ consultation of TR: modification of the protocol |
| 19-Sept-2014 | Katharina | IPG | insertion of RFC[21] | plasmid isolation from ONC/ Digestion of pORE-E3 (without XhoI and BglII-site) with MluI and BamHI/ annealing two primers (3 and 4) to build the rcf/ phosphorylation of primer construct/ dephosphorylation of digested vector/ ligation of vector and insert/ transformation of XL1-Blue Competent Cells/ incubation over night (25 °C) |
| 18-Sept-2014 | Katharina | IPG | insertion of RFC[21] | digestion of pORE-E3 (without XhoI- and BglII-site): no DNA: inoculation of an ONC |
| 17-Sept-2014 | Katharina, Björn | IPG | insertion of RFC[21] | colony-PCR with primer 1261 and 488: all 60 colonies negative: digestion probably went wrong/ isolation of plasmid of another ONC via handpreparation |
| 16-Sept-2014 | Katharina, Björn | IPG | insertion of RFC[21] | plasmid isolation from ONC/ digestion of pORE-E3 (without XhoI and BglII-site) with MluI and BamHI/ annealing two primers (3 and 4) to build the rcf/ phosphorylation of primer construct/ dephosphorylation of digested vector/ ligation of vector and insert/ transformation of XL1-Blue Competent Cells |
| 15-Sept-2014 | Katharina, Björn | IPG | insertion of RFC[21] | digestion of pORE-E3 (without XhoI and BglII-site): no DNA: inoculation of an ONC |
| 27-Aug-2014 | Melanie, Lisa | IPG | plasmidpreparation/ sequencing.v1 | plasmidpreparation with 12 colonies/ double digestion (XhoI and NcoI): 7 might be without BglII AND XhoI: sequencing with primer ??? (IPG) positive/ pORE_E3 2x35S (without T4MPB) and without BglII- and XhoI-site |
| 26-Aug-2014 | Melanie | IPG | ONC of 13 colonies (12+1 pos. control) | several colonies on each plate (including pos. control)/ pick of 3 to 4 per plate/ ONC |
| 25-Aug-2014 | Melanie, Lisa | IPG | ligation/ transformation of XL1-Blue Competent Cells via heat-shock/ selection on kanamycin | |
| 22-Aug-2014 | Melanie | IPG | digest of pORE_E3 2x35S (without T4MBP and without BglII-site) with XhoI, proofreading PCR | digestion of pORE_E3 2x35S (without T4MBP and without BglII-site) with XhoI for 1h 37 °C showed complete digestion/ proofreading-PCR to avoid A-Tailing (Phusion) |
| 20-Aug-2014 | Melanie | IPG | sequencing.v3 (new primer) | sequencing with primer 1356: positive: BglII-site destroyed |
| 19-Aug-2014 | Melanie | IPG | sequencing.v2 (same primer) | sequencing with primer 1261 (IPG) again stopped 10 bp before BglII-site: no definite result: again |
| 15-Aug-2014 | Lisa | IPG | plasmidpreparation/ sequencing.v1 | plasmidpreparation with 10 colonies/ digestion with BglI (twice): poor results: 1 probe might be without BglII: sequencing with primer 1261 (IPG) of this probe stopped 10 bp before BglII-site: no definite result: again |
| 13-Aug-2014 | Melanie, Lisa | IPG | ONC of 10 colonies (9+1 pos. control) | several colonies on each plate (including pos. control): pick of 3 per plate: ONC |
| 12-Aug-2014 | Melanie | IPG | transformation of E. coli with pORE_E3 2x35S (without T4MBP) ?without? BglII | transformation via heat-shock/ selection on kanamycin |
| 11-Aug-2014 | Melanie, Lisa | IPG | digest of pORE_E3 2x35S (without T4MBP) with BglII/ proofreading PCR/ ligation | digestion of pORE_E3 2x35S (without T4MPB) with BglII for 1h 37 °C: complete digestion/ proofreading-PCR to avoid A-Tailing (Phusion)/ ligation |
