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Team:Hong Kong HKUST/wetlab/notebook
From 2014.igem.org
(Difference between revisions)
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<a href="#" class="head">Week 2</a> | <a href="#" class="head">Week 2</a> | ||
<div class="content" align="left" style="display: none;"> | <div class="content" align="left" style="display: none;"> | ||
| - | <br><b>June | + | <br><b>June 9</b> <br> |
Wet lab<br> | Wet lab<br> | ||
| + | · Transformation of pSB1C3-BBa_E0240, pSB1C3-BBa_B0032 and pSB1C3-BBa_K20600<br> | ||
· <br> | · <br> | ||
· <br> | · <br> | ||
| + | · <br> | ||
| + | <br><b>June 10</b> <br> | ||
| + | Wet lab<br> | ||
| + | · Found reference promoter J23101 in BBa_I20260 from part registry<br> | ||
| + | · Looked for Pcombox gene sequence from the genomic DNA<br> | ||
· <br> | · <br> | ||
· <br> | · <br> | ||
| - | <br><b>June | + | <br><b>June 11</b> <br> |
Wet lab<br> | Wet lab<br> | ||
| - | · <br> | + | · Looked for Pcombox gene sequence from the genomic DNA<br> |
| - | · <br> | + | · Studied the Characteristic, function and mechnism of how promoters work<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
| - | <br><b>June | + | <br><b>June 12</b> <br> |
Wet lab<br> | Wet lab<br> | ||
| - | · <br> | + | · Looked for Pcombox gene sequence from the genomic DNA<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
· <br> | · <br> | ||
| - | <br><b>June | + | <br><b>June 13</b> <br> |
Wet lab<br> | Wet lab<br> | ||
| - | · <br> | + | · Looked for Pcombox gene sequence from the genomic DNA<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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<a href="#" class="head">Week 3</a> | <a href="#" class="head">Week 3</a> | ||
<div class="content" align="left" style="display: none;"> | <div class="content" align="left" style="display: none;"> | ||
| - | <br><b>June | + | |
| + | <br><b>June 16</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
| - | · <br> | + | · Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for Pcombox gene sequence<br> |
| - | · <br> | + | · Found a possible Pcombox<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
| - | <br><b>June | + | <br><b>June 17</b> <br> |
Wet lab<br> | Wet lab<br> | ||
| - | · <br> | + | · Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for Pcombox gene sequence<br> |
| - | · <br> | + | · Found a possible Pcombox<br> |
| - | · <br> | + | · Searched for other possible Pcombox sequences<br> |
· <br> | · <br> | ||
| - | <br><b>June | + | <br><b>June 18</b> <br> |
Wet lab<br> | Wet lab<br> | ||
| + | · Designed primers for the Pcombox sequences<br> | ||
| + | · Received a Pcombox sequence from Wellcome Trust Sanger Institute<br> | ||
· <br> | · <br> | ||
| - | · | + | <br><b>June 19</b> <br> |
| - | · | + | · Digestion of pSB1C3-BBa_E0240 at EcoRI and SpeI<br> |
| - | · | + | · <br> |
| - | <br><b>June | + | · <br> |
| - | + | · <br> | |
| - | · | + | <br><b>June 20</b> <br> |
| - | · | + | · Amended primers of Pcombox sequences<br> |
| - | · | + | · <br> |
| - | · | + | · <br> |
| + | · <br> | ||
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· Transformation of BBa_B0015 from iGEM Kit Plate 2011 and BBa_K880005<br> | · Transformation of BBa_B0015 from iGEM Kit Plate 2011 and BBa_K880005<br> | ||
· 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> | ||
| - | · <br> | + | · Designed plasmid construct for Pcombox<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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Wet lab<br> | Wet lab<br> | ||
· Inoculation of BBa_B0015<br> | · Inoculation of BBa_B0015<br> | ||
| - | · <br> | + | · Designed new reverse primer for 100bp, 150bp, 160bp, 180bp and 249bp Pcombox<br> |
| - | · <br> | + | · Designed forward and reverse oligos of 67bp Pcombox<br> |
| - | · <br> | + | · Transformation of pSB1C3-BBa_I13504<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification<br> | · Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification<br> | ||
· Inoculation of BBa_K880005<br> | · Inoculation of BBa_K880005<br> | ||
| - | · <br> | + | · Inoculation of pSB1C3-BBa_E0240<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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Wet lab<br> | Wet lab<br> | ||
· 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> | ||
| - | · <br> | + | · Digestion of pSB1C3-BBa_E0240 at XbaI and PstI<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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Wet lab<br> | Wet lab<br> | ||
· Ligation of BBa_B0015 insert cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI as negative control<br> | · Ligation of BBa_B0015 insert cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI as negative control<br> | ||
| - | · <br> | + | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> |
| - | · <br> | + | · Gel purification of pSB1C3-BBa_E0240<br> |
· <br> | · <br> | ||
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· Dilution of primers and design of experiment for both comX-myc and comW-FLAG<br> | · Dilution of primers and design of experiment for both comX-myc and comW-FLAG<br> | ||
· Inoculation of ligated pSB1C3-K880005.B0015<br> | · Inoculation of ligated pSB1C3-K880005.B0015<br> | ||
| - | · <br> | + | · Inoculation of pSB3K3-BBa_J04450 and pSB1C3-BBa_E0240<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Gel check for comX-myc PCR product (no PCR product present)<br> | · Gel check for comX-myc PCR product (no PCR product present)<br> | ||
· Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 with XbaI and PstI<br> | · Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 with XbaI and PstI<br> | ||
| - | · <br> | + | · Received and stored the new common reverse primers for Pcombox and sense oligo and anti-sense oligo of 67bp Pcombox<br> |
| - | · <br> | + | · Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br> |
| - | · <br> | + | · Gel purification of digested BBa_E0240 and BBa_J04450<br> |
| + | · Inoculation of pSB3K3-BBa_J04450<br> | ||
| + | · <br> | ||
| + | · <br> | ||
· <br> | · <br> | ||
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Wet lab<br> | Wet lab<br> | ||
· PCR of comX-myc using Vent DNA Polymerase, PCR clean up<br> | · PCR of comX-myc using Vent DNA Polymerase, PCR clean up<br> | ||
| - | · <br> | + | · Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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</div> | </div> | ||
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· Gel check for comW-FLAG PCR product, PCR clean up<br> | · Gel check for comW-FLAG PCR product, PCR clean up<br> | ||
· Dephosporylation of digested pSB1C3-K880005 backbone cut with SpeI and PstI<br> | · Dephosporylation of digested pSB1C3-K880005 backbone cut with SpeI and PstI<br> | ||
| - | · <br> | + | · Gel purification of digested BBa_E0240 and BBa_J04450<br> |
| - | · <br> | + | · Submitted primers for Gibson assembly of pSB3K3-100bpPcombox<br> |
| - | · <br> | + | · Inoculation of pSB3K3-BBa_J04450<br> |
| + | · <br> | ||
| + | · <br> | ||
· <br> | · <br> | ||
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· 2nd attempt of ligation of BBa_B0015 insert cut with XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut with SpeI and PstI as negative control<br> | · 2nd attempt of ligation of BBa_B0015 insert cut with XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut with SpeI and PstI as negative control<br> | ||
· Transformation of ligation products<br> | · Transformation of ligation products<br> | ||
| - | · <br> | + | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> |
| - | · <br> | + | · Gel purification of digested BBa_J04450<br> |
| - | · <br> | + | · Received pCEPcin from Universite de Toulouse, France<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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Wet lab<br> | Wet lab<br> | ||
· Inoculation of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br> | · Inoculation of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br> | ||
| - | · <br> | + | · Ethanol precipitation of pCEPcin<br> |
| - | · <br> | + | · Gel purification of digested BBa_E0240<br> |
| - | · <br> | + | · Gel check for BBa_E0240 insert and pSB3K3 backbone<br> |
| - | · <br> | + | · PCR of 100bp Pcombox using Phusion polymerase<br> |
| - | · <br> | + | · Inoculation of pCEPcin<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
· <br> | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | |||
| + | |||
<br> <b> July 10</b> <br> | <br> <b> July 10</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
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· Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br> | · Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br> | ||
· Gel check and insert wanted present in several candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt), but no insert wanted present in any candidate colonies of ligated pSB1C3-K880005.comX-myc<br> | · Gel check and insert wanted present in several candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt), but no insert wanted present in any candidate colonies of ligated pSB1C3-K880005.comX-myc<br> | ||
| - | · <br> | + | · Digestion of pCEPcin at EcoRI and SpeI, Xho and BamHI<br> |
| - | · <br> | + | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> |
| + | · PCR of 100bp Pcombox using Phusion polymerase<br> | ||
| + | · Ethanol precipitation of BBa_E0240 insert<br> | ||
· <br> | · <br> | ||
· <br> | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | |||
<br> <b> July 11</b> <br> | <br> <b> July 11</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
· Digestion of comW-FLAG with XbaI and PstI, digestion clean up<br> | · Digestion of comW-FLAG with XbaI and PstI, digestion clean up<br> | ||
| - | · <br> | + | · Gel check for the PCR product obtained on July 10<br> |
| - | · <br> | + | · Digestion of pCEPcin at NcoI and XhoI<br> |
| - | · <br> | + | · Ethanol precipitation of pCEPcin<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Transformation of ligation products<br> | · Transformation of ligation products<br> | ||
· Inoculation of ligated dephosporylated pSB1C3-K880005.comX-myc<br> | · Inoculation of ligated dephosporylated pSB1C3-K880005.comX-myc<br> | ||
| - | · <br> | + | · Digestion of pCEPcin at NcoI and XhoI<br> |
| - | · <br> | + | · PCR of 100bp Pcombox using Phusion polymerase<br> |
| - | · <br> | + | · Gel purification of digested BBa_J04450<br> |
| - | · <br> | + | · Inoculation of pCEPcin<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Gel check of pSB1C3-K880005.comX-myc PCR product (no PCR product present)<br> | · Gel check of pSB1C3-K880005.comX-myc PCR product (no PCR product present)<br> | ||
· Inoculation of dephosporylated pSB1C3-K880005.comX-myc, dephosporylated pSB1C3-K880005.comW-FLAG<br> | · Inoculation of dephosporylated pSB1C3-K880005.comX-myc, dephosporylated pSB1C3-K880005.comW-FLAG<br> | ||
| + | · Ethanol precipitation of pCEPcin<br> | ||
| + | · Gel check of the PCR product obtained on July 14<br> | ||
| + | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
| + | · PCR of 150bp, 160bp, 180bp, 249bp and 300bp Pcombox using Phusion polymerase<br> | ||
| + | · <br> | ||
· <br> | · <br> | ||
| + | · <br> | ||
· <br> | · <br> | ||
· <br> | · <br> | ||
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· PCR of comW using Vent DNA Polymerase (no PCR product present)<br> | · PCR of comW using Vent DNA Polymerase (no PCR product present)<br> | ||
· Digestion of comX on XbaI and PstI<br> | · Digestion of comX on XbaI and PstI<br> | ||
| - | · <br> | + | · Gel check of the PCR product obtained on July 15<br> |
| - | · <br> | + | · Gel purification of digested pSB3K3 backbone<br> |
| - | · <br> | + | · Digestion of pCEPcin2 at XhoI and SalI<br> |
| + | · PCR of 150bp, 160bp, 180bp, 249bp and 300bp Pcombox using Phusion polymerase<br> | ||
| + | · Inoculation of pCEPcin2 and pSB3K3-BBa_I20260<br> | ||
| + | · <br> | ||
| + | · <br> | ||
· <br> | · <br> | ||
| + | |||
<br> <b> July 17</b> <br> | <br> <b> July 17</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
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· Gel extraction and gel purification of comW PCR product<br> | · Gel extraction and gel purification of comW PCR product<br> | ||
· Dephosporylation of BBa_K880005<br> | · Dephosporylation of BBa_K880005<br> | ||
| - | · <br> | + | · Gel check of the PCR product obtained on July 16, all types of Pcombox shown positive result<br> |
| - | · <br> | + | · PCR clean-up of Pcombox<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
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· Digestion of comX-myc and comW PCR products, digestion clean-up<br> | · Digestion of comX-myc and comW PCR products, digestion clean-up<br> | ||
· Gel extraction and gel purification of digested comX and comX-myc<br> | · Gel extraction and gel purification of digested comX and comX-myc<br> | ||
| + | · Digestion of pCEPcin2 (at XhoI and SalI) and pSB3K3-BBa_I20260 (at XbaI and PstI)<br> | ||
| + | · Digestion of pCEPcin2 (at BamHI and NcoI)<br> | ||
| + | · PCR of 100bp Pcombox using Phusion polymerase<br> | ||
| + | · Gel check of the purified Pcombox obtained July 17<br> | ||
| + | · Gel check of the 100bp Pcombox (PCR product)<br> | ||
| + | · Gel purification of digested pSB3K3 backbone<br> | ||
| + | · <br> | ||
· <br> | · <br> | ||
| + | · <br> | ||
· <br> | · <br> | ||
· <br> | · <br> | ||
| + | |||
</div> | </div> | ||
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· Ligation of comW into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br> | · Ligation of comW into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br> | ||
· Colony PCR of pSB1C3-K880005.comW-FLAG using Taq DNA Polymerase<br> | · Colony PCR of pSB1C3-K880005.comW-FLAG using Taq DNA Polymerase<br> | ||
| - | · <br> | + | · PCR of 100bp and 150bp Pcombox using Phusion polymerase<br> |
| - | · <br> | + | · Gel purification of digested pSB3K3 backbone<br> |
| + | · <br> | ||
· <br> | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | |||
<br> <b> July 22</b> <br> | <br> <b> July 22</b> <br> | ||
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· Colony PCR of comX, comW, comX-myc, comW-FLAG and use BBa_J04450 as the positive control using Taq DNA Polymerase (no PCR product present)<br> | · Colony PCR of comX, comW, comX-myc, comW-FLAG and use BBa_J04450 as the positive control using Taq DNA Polymerase (no PCR product present)<br> | ||
· Inoculation of comX, comW, comX-myc, comW-FLAG<br> | · Inoculation of comX, comW, comX-myc, comW-FLAG<br> | ||
| - | · <br> | + | · Gel check of the PCR product obtained on July 21<br> |
| - | · <br> | + | · Oligo annealing of 67bp Pcombox<br> |
| - | · <br> | + | · Digestion of pSB3K3-BBa_I20260 at XbaI and PstI<br> |
| - | · <br> | + | · PCR of 100bp,150bp and 160bp Pcombox using Phusion polymerase<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
| Line 492: | Line 519: | ||
· Digestion check of comX, comW, comX-myc, comW-FLAG colony PCR products on XbaI and SpeI<br> | · Digestion check of comX, comW, comX-myc, comW-FLAG colony PCR products on XbaI and SpeI<br> | ||
· Inoculation of comW-FLAG<br> | · Inoculation of comW-FLAG<br> | ||
| + | · Gel check of PCR product obtained on July 22<br> | ||
| + | · Gel purification of digested pSB3K3 backbone<br> | ||
| + | · PCR of 100bp,150bp and 160bp Pcombox using Phusion polymerase<br> | ||
| + | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> | ||
| + | · Gel purification of digested pSB3K3 backbone<br> | ||
· <br> | · <br> | ||
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| - | |||
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· <br> | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
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<br> <b> July 24</b> <br> | <br> <b> July 24</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
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· PCR of comX-myc and comX using Vent DNA Polymerase, gel extraction and gel purification<br> | · PCR of comX-myc and comX using Vent DNA Polymerase, gel extraction and gel purification<br> | ||
· Digestion of comX-myc and comX on XbaI and PstI-HF<br> | · Digestion of comX-myc and comX on XbaI and PstI-HF<br> | ||
| - | · <br> | + | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> |
| - | · <br> | + | · Gel Check of PCR product obtained on July 23<br> |
| - | · <br> | + | · Received pCEPcin2<br> |
| + | · <br> | ||
· <br> | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | · <br> | ||
| + | |||
<br> <b> July 25</b> <br> | <br> <b> July 25</b> <br> | ||
Wet lab<br> | Wet lab<br> | ||
· Colony PCR of comX-myc using Taq DNA Polymerase<br> | · Colony PCR of comX-myc using Taq DNA Polymerase<br> | ||
· Gel check of comX-myc PCR product (no PCR product present)<br> | · Gel check of comX-myc PCR product (no PCR product present)<br> | ||
| - | · <br> | + | · Gel purification of digested pSB3K3 backbone<br> |
| - | · <br> | + | · Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br> |
· <br> | · <br> | ||
· <br> | · <br> | ||
Revision as of 12:19, 10 October 2014
Notebook
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