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Team:Hong Kong HKUST/wetlab/notebook
From 2014.igem.org
(Difference between revisions)
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· Designed primers for the Pcombox sequences<br> | · Designed primers for the Pcombox sequences<br> | ||
· Received a Pcombox sequence from Wellcome Trust Sanger Institute<br> | · Received a Pcombox sequence from Wellcome Trust Sanger Institute<br> | ||
| + | · Miniprep of BBa_J04450(CHL) and BBa_I20260<br> | ||
| + | · Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260<br> | ||
| + | · Digestion of BBa_J04450(CHL) and BBa_I20260<br> | ||
| + | · Gel electrophoresis of digested products<br> | ||
· <br> | · <br> | ||
<br><b>June 19</b> <br> | <br><b>June 19</b> <br> | ||
· Digestion of pSB1C3-BBa_E0240 at EcoRI and SpeI<br> | · Digestion of pSB1C3-BBa_E0240 at EcoRI and SpeI<br> | ||
| - | · <br> | + | · Miniprep of BBa_J04450(CHL) and BBa_I20260<br> |
| - | · <br> | + | · Digestion of BBa_J04450(CHL)<br> |
| + | · Gel electrophoresis of digested product<br> | ||
| + | · Bacterial inoculation of BBa_J04450(CHL)<br> | ||
· <br> | · <br> | ||
<br><b>June 20</b> <br> | <br><b>June 20</b> <br> | ||
· Amended primers of Pcombox sequences<br> | · Amended primers of Pcombox sequences<br> | ||
| - | · <br> | + | · Miniprep of BBa_J04450(CHL)<br> |
| - | · <br> | + | · Digestion of BBa_J04450(CHL)<br> |
| - | · <br> | + | · Gel electrophoresis of digested product<br> |
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· Primer design for PCR of comX-myc and comW-FLAG from genomic DNA of S. Pneumoniae NCTC 7465 strain<br> | · Primer design for PCR of comX-myc and comW-FLAG from genomic DNA of S. Pneumoniae NCTC 7465 strain<br> | ||
· Designed plasmid construct for Pcombox<br> | · Designed plasmid construct for Pcombox<br> | ||
| - | · <br> | + | · Bacterial inoculation of BBa_J04450(CHL)<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Designed forward and reverse oligos of 67bp Pcombox<br> | · Designed forward and reverse oligos of 67bp Pcombox<br> | ||
· Transformation of pSB1C3-BBa_I13504<br> | · Transformation of pSB1C3-BBa_I13504<br> | ||
| - | · <br> | + | · Miniprep of BBa_J04450(CHL)<br> |
| - | · <br> | + | · Transformation of BBa_J04450(KAN)<br> |
| - | · <br> | + | · Bacterial inoculation of BBa_E0240<br> |
· <br> | · <br> | ||
<br><b>June 25</b> <br> | <br><b>June 25</b> <br> | ||
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· Inoculation of BBa_K880005<br> | · Inoculation of BBa_K880005<br> | ||
· Inoculation of pSB1C3-BBa_E0240<br> | · Inoculation of pSB1C3-BBa_E0240<br> | ||
| - | · <br> | + | · Miniprep of BBa_E0240<br> |
| - | · <br> | + | · Digestion of BBa_E0240<br> |
| - | · <br> | + | · Transformation of BBa_J04450(KAN)<br> |
| - | · <br> | + | · Bacterial inoculation of BBa_J04450(CHL)<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
| Line 243: | Line 249: | ||
· Digestion of BBa_K880005 plasmid extraction product with SpeI and PstI, gel extraction and gel purification<br> | · Digestion of BBa_K880005 plasmid extraction product with SpeI and PstI, gel extraction and gel purification<br> | ||
· Digestion of pSB1C3-BBa_E0240 at XbaI and PstI<br> | · Digestion of pSB1C3-BBa_E0240 at XbaI and PstI<br> | ||
| - | · <br> | + | · Gel electrophoresis of digested BBa_E0240<br> |
| + | · Gel extraction and purification of digested BBa_E0240 to obtain the insert<br> | ||
| + | · Miniprep of BBa_J04450(CHL)<br> | ||
| + | · Digestion of BBa_J04450(CHL)<br> | ||
| + | · Gel electrophoresis of digested BBa_J04450(CHL)<br> | ||
| + | · Gel extraction and purification of digested BBa_J04450(CHL) to obtain pSB1C3<br> | ||
| + | · Bacterial inoculation of BBa_K880005<br> | ||
· <br> | · <br> | ||
<br><b>June 27</b> <br> | <br><b>June 27</b> <br> | ||
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· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
· Gel purification of pSB1C3-BBa_E0240<br> | · Gel purification of pSB1C3-BBa_E0240<br> | ||
| + | · Miniprep of BBa_K880005<br> | ||
· <br> | · <br> | ||
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· Inoculation of ligated pSB1C3-K880005.B0015<br> | · Inoculation of ligated pSB1C3-K880005.B0015<br> | ||
· Inoculation of pSB3K3-BBa_J04450 and pSB1C3-BBa_E0240<br> | · Inoculation of pSB3K3-BBa_J04450 and pSB1C3-BBa_E0240<br> | ||
| - | · <br> | + | · Oligo annealing for PcomX<br> |
| + | · Bacterial inoculation of BBa_J04450 (KAN) (6 tubes), BBa_E0240 (CAM) (6 tubes), BBa_B0015 (KAN) (3 tubes)<br> | ||
· <br> | · <br> | ||
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· Gel purification of digested BBa_E0240 and BBa_J04450<br> | · Gel purification of digested BBa_E0240 and BBa_J04450<br> | ||
· Inoculation of pSB3K3-BBa_J04450<br> | · Inoculation of pSB3K3-BBa_J04450<br> | ||
| - | · | + | · PcomX Transformation<br> |
| - | · | + | · PcomCDE Oligo annealing, ligation, transformation<br> |
| - | · | + | · J04450 Plasmid Extraction<br> |
| + | · E0240:Plasmid extraction (6 Falcon tubes); Nanodrop; Digestion by XbaI and SpeI, CutSmart as Buffer (only do 3 tubes); Gel electrophoresis; Gel extraction (INSERT!!! FRONT band); Gel purification<br> | ||
| + | · B0015: Plasmid extraction (3 Falcon tubes); Nanodrop<br> | ||
| + | · comED58E: PCR<br> | ||
| + | · <br> | ||
<br> <b> July 4</b> <br> | <br> <b> July 4</b> <br> | ||
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· PCR of comX-myc using Vent DNA Polymerase, PCR clean up<br> | · PCR of comX-myc using Vent DNA Polymerase, PCR clean up<br> | ||
· Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
| - | · <br> | + | · comED58E :Phusion PCR<br> |
| - | · <br> | + | · E0240: Gel purification<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Submitted primers for Gibson assembly of pSB3K3-100bpPcombox<br> | · Submitted primers for Gibson assembly of pSB3K3-100bpPcombox<br> | ||
· Inoculation of pSB3K3-BBa_J04450<br> | · Inoculation of pSB3K3-BBa_J04450<br> | ||
| - | · | + | · E0240 - Bacterial inoculation<br> |
| - | · | + | · pSB1C3-PcomX: Bacterial inoculation<br> |
| + | · pSB1C3- PcomCDE: Bacterial inoculation, Streaking<br> | ||
| + | · comED58E :PCR cleanup; Digestion with EcoRI and PstI; Ligation with pSB1C3; Transformation<br> | ||
· <br> | · <br> | ||
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· Gel purification of digested BBa_J04450<br> | · Gel purification of digested BBa_J04450<br> | ||
· Received pCEPcin from Universite de Toulouse, France<br> | · Received pCEPcin from Universite de Toulouse, France<br> | ||
| - | · <br> | + | · J04450 (KAN): Digestion by EcoRI and PstI; Gel electrophoresis; Gel extraction<br> |
| - | · <br> | + | · E0240: Plasmid extraction<br> |
| + | · pSB1C3-PcomX: Plasmid extraction<br> | ||
| + | · pSB1C3- PcomCDE: Plasmid extraction<br> | ||
| + | · comED58E: Bacterial inoculation; Ligation (redo); Transformation (redo)<br> | ||
· <br> | · <br> | ||
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· PCR of 100bp Pcombox using Phusion polymerase<br> | · PCR of 100bp Pcombox using Phusion polymerase<br> | ||
· Inoculation of pCEPcin<br> | · Inoculation of pCEPcin<br> | ||
| - | · <br> | + | · E0240-PcomX & PcomCDE: Digestion w/ EcoRI and PstI; Gel electrophoresis<br> |
| - | · <br> | + | · J04450 (KAN): Gel purification; NanoDrop (low conc)<br> |
| - | · | + | · comED58E:Plasmid extraction; NanoDrop; Gel electrophoresis (not comE); Bacterial inoculation (redo)July<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· PCR of 100bp Pcombox using Phusion polymerase<br> | · PCR of 100bp Pcombox using Phusion polymerase<br> | ||
· Ethanol precipitation of BBa_E0240 insert<br> | · Ethanol precipitation of BBa_E0240 insert<br> | ||
| - | · <br> | + | · Ligate E0240-PcomX & PcomCDE <br> |
| - | · | + | · Extract J04450 (KAN)<br> |
| - | · | + | · Restrict check & run mutagenesis comED58E<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Digestion of pCEPcin at NcoI and XhoI<br> | · Digestion of pCEPcin at NcoI and XhoI<br> | ||
· Ethanol precipitation of pCEPcin<br> | · Ethanol precipitation of pCEPcin<br> | ||
| - | · <br> | + | · Extract J04450 (KAN)<br> |
| - | · <br> | + | · Ligate pSB1C3 with comED58E <br> |
| - | · <br> | + | · TransformPcomCDE<br> |
· <br> | · <br> | ||
</div> | </div> | ||
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· Gel purification of digested BBa_J04450<br> | · Gel purification of digested BBa_J04450<br> | ||
· Inoculation of pCEPcin<br> | · Inoculation of pCEPcin<br> | ||
| - | · <br> | + | · PCR of comED58E with only 1 primer → check whether the primers are working or not<br> |
| + | · Ligation of pSB1C3-PcomX<br> | ||
| + | · Inoculation of pSB1C3-PcomCDE<br> | ||
| + | · Digest B0015 w/ NotI and SpeI stick ends<br> | ||
| + | · Digest comED58E w/ XbaI and NotI sticky ends:<br> | ||
· <br> | · <br> | ||
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· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
· PCR of 150bp, 160bp, 180bp, 249bp and 300bp Pcombox using Phusion polymerase<br> | · PCR of 150bp, 160bp, 180bp, 249bp and 300bp Pcombox using Phusion polymerase<br> | ||
| - | · | + | · Plasmid extraction of pSB1C3-PcomCDE<br> |
| - | · <br> | + | · Bacterial inoculation (5 for PcomCDE, 4 for PcomX)<br> |
| - | · | + | · Bacterial inoculation of B0015 (6 tubes)<br> |
| - | · <br> | + | · Extract gel B0015 & comED58E<br> |
| + | · K880005 dephosphorylation; Digestion with SpeI<br> | ||
· <br> | · <br> | ||
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· PCR of 150bp, 160bp, 180bp, 249bp and 300bp Pcombox using Phusion polymerase<br> | · PCR of 150bp, 160bp, 180bp, 249bp and 300bp Pcombox using Phusion polymerase<br> | ||
· Inoculation of pCEPcin2 and pSB3K3-BBa_I20260<br> | · Inoculation of pCEPcin2 and pSB3K3-BBa_I20260<br> | ||
| - | · | + | · Plasmid extraction of Promoters and B0015<br> |
| - | · | + | · Colony PCR<br> |
| + | · Extract gel B0015 & comED58E by XbaI (comE) and SpeI (B0015)<br> | ||
· <br> | · <br> | ||
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· Gel check of the PCR product obtained on July 16, all types of Pcombox shown positive result<br> | · Gel check of the PCR product obtained on July 16, all types of Pcombox shown positive result<br> | ||
· PCR clean-up of Pcombox<br> | · PCR clean-up of Pcombox<br> | ||
| - | · <br> | + | · PCR product ligation with pSB1C3<br> |
| + | · Bacterial inoculation of B0015, comED58E<br> | ||
| + | · Extract gel B0015 & comED58E - Digestion by XbaI (comED58E) and SpeI (B0015)<br> | ||
| + | · Overnight Ligation<br> | ||
· <br> | · <br> | ||
<br> <b> July 18</b> <br> | <br> <b> July 18</b> <br> | ||
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· Gel check of the 100bp Pcombox (PCR product)<br> | · Gel check of the 100bp Pcombox (PCR product)<br> | ||
· Gel purification of digested pSB3K3 backbone<br> | · Gel purification of digested pSB3K3 backbone<br> | ||
| - | · | + | · Ligation of comED58E<br> |
| - | · <br> | + | · Gel extract comED58E<br> |
| - | · | + | · B0015 Gel purification<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· PCR of 100bp and 150bp Pcombox using Phusion polymerase<br> | · PCR of 100bp and 150bp Pcombox using Phusion polymerase<br> | ||
· Gel purification of digested pSB3K3 backbone<br> | · Gel purification of digested pSB3K3 backbone<br> | ||
| - | · | + | · Colony PCR of Promoters<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Digestion of pSB3K3-BBa_I20260 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_I20260 at XbaI and PstI<br> | ||
· PCR of 100bp,150bp and 160bp Pcombox using Phusion polymerase<br> | · PCR of 100bp,150bp and 160bp Pcombox using Phusion polymerase<br> | ||
| - | · <br> | + | · Gel electrophoresis of colony PCR results<br> |
| - | · <br> | + | · Mutagenesis of comED58E<br> |
· <br> | · <br> | ||
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· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
· Gel purification of digested pSB3K3 backbone<br> | · Gel purification of digested pSB3K3 backbone<br> | ||
| - | · <br> | + | · Gel electrophoresis of colony PCR results<br> |
| - | · | + | · Mutagenesis of comED58E<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Gel Check of PCR product obtained on July 23<br> | · Gel Check of PCR product obtained on July 23<br> | ||
· Received pCEPcin2<br> | · Received pCEPcin2<br> | ||
| - | · | + | · Gel electrophoresis of colony PCR and mutagenesis results<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Gel purification of digested pSB3K3 backbone<br> | · Gel purification of digested pSB3K3 backbone<br> | ||
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
| - | · <br> | + | · Gel check on K880005<br> |
| - | · <br> | + | · Transformation of ligated product (promoters+pSB1C3)<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
Revision as of 16:08, 16 October 2014
Notebook
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Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and research findings. The notebook for Pneuumonsensor and Riboregulator are displayed separately. PneumosensorRiboregulator |
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