Team:Hong Kong HKUST/wetlab/notebook

From 2014.igem.org

(Difference between revisions)
Line 609: Line 609:
·      Inoculation of comX-myc and comW-FLAG<br>
·      Inoculation of comX-myc and comW-FLAG<br>
·      Digestion of pCEPcin2 at EcoRI and PstI, EcoRI and SpeI, NcoI and XhoI<br>
·      Digestion of pCEPcin2 at EcoRI and PstI, EcoRI and SpeI, NcoI and XhoI<br>
-
·      <br>
+
·      Colony PCR of promoters-pSB1C3<br>
 +
·      Bacterial inoculation of K880005, comED58E <br>
·      <br>
·      <br>
·   <br>
·   <br>
Line 621: Line 622:
·      Inoculation of pSB3K3-BBa_J04450<br>
·      Inoculation of pSB3K3-BBa_J04450<br>
·      PCR of 100bp,150bp and 160bp Pcombox using Vent polymerase<br>
·      PCR of 100bp,150bp and 160bp Pcombox using Vent polymerase<br>
-
·   <br>
+
·     Restrict check K880005 <br>
-
·      <br>
+
·      Promoters colony PCR <br>
·      <br>
·      <br>
·      <br>
·      <br>
Line 635: Line 636:
·      Digestion of pCEPcin2 at EcoRI and PstI<br>
·      Digestion of pCEPcin2 at EcoRI and PstI<br>
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
-
·   <br>
+
·     Restrict check K880005  <br>
<br> <b> July 31</b> <br>
<br> <b> July 31</b> <br>
Wet lab<br>
Wet lab<br>
Line 643: Line 644:
·      Ethanol precipitation for comW and comX<br>
·      Ethanol precipitation for comW and comX<br>
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
-
·      <br>
+
·      K880005 dephosphorylation & ligation<br>
-
·   <br>
+
·     Promoters Colony PCR + Sequencing reaction  <br>
       </div>
       </div>
Line 669: Line 670:
Wet lab<br>
Wet lab<br>
·      Digestion of pSB1C3-BBa_J04450<br>
·      Digestion of pSB1C3-BBa_J04450<br>
-
·      <br>
+
·      Restrict check Promoters & Sequencing reaction<br>
-
·      <br>
+
·      Transform K880005<br>
·   <br>
·   <br>
Line 690: Line 691:
·      Ligation of 100bp and 160bp Pcombox with pSB1C3<br>
·      Ligation of 100bp and 160bp Pcombox with pSB1C3<br>
·   Digestion of pCEPcin2 at EcoRI and PstI<br>
·   Digestion of pCEPcin2 at EcoRI and PstI<br>
-
·      <br>
+
·      PcomCDE Precipitation<br>
-
·      <br>
+
·      PcomX Sequencing reaction & precipitation<br>
-
·   <br>
+
·     Transform comED58E  <br>
·      <br>
·      <br>
·      <br>
·      <br>
Line 709: Line 710:
·      Gel purification of digested pSB1C3 backbone<br>
·      Gel purification of digested pSB1C3 backbone<br>
·   Inoculation of pCEPcin2<br>
·   Inoculation of pCEPcin2<br>
-
·      <br>
+
·      Send to sequencing: PcomCDE and PcomX<br>
-
·   <br>
+
·     Bacterial inoculation of comED58E  <br>
·   <br>
·   <br>
·      <br>
·      <br>
Line 727: Line 728:
·      Oligo annealing for 67bp Pcombox<br>
·      Oligo annealing for 67bp Pcombox<br>
·   <br>
·   <br>
-
·      <br>
+
·      Align sequence with expected: PcomCDE and Pcom<br>
·      <br>
·      <br>
·      <br>
·      <br>
Line 740: Line 741:
·      Colony PCR of 100bp, 150bp, 160bp, 180bp and 249bp Pcombox<br>
·      Colony PCR of 100bp, 150bp, 160bp, 180bp and 249bp Pcombox<br>
·      Gel purification of digested pSB1C3 backbone<br>
·      Gel purification of digested pSB1C3 backbone<br>
-
·      <br>
+
·      Precipitate after sequencing reaction PcomCDE<br>
-
·      <br>
+
·      Make glycerol stock for PcomX<br>
-
·      <br>
+
·      Run colony PCR for comED58E<br>
·   <br>
·   <br>
<br><b>Aug 8</b> <br>
<br><b>Aug 8</b> <br>
Line 755: Line 756:
·      Digestion of pCEPcin2 at EcoRI and EcoRV, XhoI and NcoI<br>
·      Digestion of pCEPcin2 at EcoRI and EcoRV, XhoI and NcoI<br>
·      Ligation of 67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp Pcombox with pSB1C3 backbone<br>
·      Ligation of 67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp Pcombox with pSB1C3 backbone<br>
-
·   <br>
+
·     Ligate with E0240 PcomX  <br>
-
·      <br>
+
·      Colony PCR for comE mutant<br>
-
·      <br>
+
·      Check sequence PcomCDE<br>
·   <br>
·   <br>
Line 774: Line 775:
·      Gel purification of digested pSB1C3 backbone<br>
·      Gel purification of digested pSB1C3 backbone<br>
·   Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
·   Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
-
·      <br>
+
·      Extract pSB1C3 from J04450<br>
-
·      <br>
+
·      Redo ligation part 1<br>
-
·      <br>
+
·      Gibson assembly of comED58E<br>
·   <br>
·   <br>
<br><b>Aug 12</b> <br>
<br><b>Aug 12</b> <br>
Line 788: Line 789:
·      Digestion of pCEPcin2 at EcoRI and EcoRV<br>
·      Digestion of pCEPcin2 at EcoRI and EcoRV<br>
·      Restriction check of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br>
·      Restriction check of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br>
-
·      <br>
+
·      Ligate K880005 and comE(D58E)<br>
 +
·      Inoculate for glycerol stock (PcomX)<br>
·   <br>
·   <br>
<br><b>Aug 13</b> <br>
<br><b>Aug 13</b> <br>
Line 796: Line 798:
·      Restriction check of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br>
·      Restriction check of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br>
·      Colony PCR of pSB1C3-Pcombox (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br>
·      Colony PCR of pSB1C3-Pcombox (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br>
-
·      <br>
+
·      Check new dNTP<br>
-
·      <br>
+
·      Restrict check of ligation product part 1<br>
-
·      <br>
+
·      Colony PCR for checking part 2 ligation product and gibson assembly<br>
-
·      <br>
+
·      Inoculation of PcomX for glycerol stock<br>
·      <br>
·      <br>
·   <br>
·   <br>
Line 812: Line 814:
·      Gel check of the digested pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) obtained on July 13<br>
·      Gel check of the digested pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp) obtained on July 13<br>
·      Colony PCR of Colony PCR of pSB1C3-Pcombox (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br>
·      Colony PCR of Colony PCR of pSB1C3-Pcombox (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br>
-
·      <br>
+
·      glycerol stock for PcomX<br>
 +
·      colony PCR ligation product part 1<br>
 +
·      colony PCR gibson assembly<br>
 +
·      Run gel for PCR products<br>
 +
·      Inoculation of comED58E<br>
·   <br>
·   <br>
<br><b>Aug 15</b> <br>
<br><b>Aug 15</b> <br>
Line 828: Line 834:
·      Preparation for submittion of samples to be sequenced<br>
·      Preparation for submittion of samples to be sequenced<br>
·      Inoculation of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp)<br>
·      Inoculation of pSB1C3-Pcombox (100bp, 150bp, 160bp, 180bp and 249bp)<br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
·      <br>
·      <br>
·      <br>
·      <br>
Line 2,099: Line 2,108:
. Order new TA and CR <br>
. Order new TA and CR <br>
. Gibson Assembly mastermix <br>
. Gibson Assembly mastermix <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
<br><b>Aug 16</b> <br>
<br><b>Aug 16</b> <br>
Line 2,104: Line 2,117:
. Miniprep <br>
. Miniprep <br>
. Colony PCR for final constructs <br>
. Colony PCR for final constructs <br>
-
 
+
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
Line 2,132: Line 2,148:
. Prepare for Pbad characterization <br>
. Prepare for Pbad characterization <br>
. Take over the tank B and C arround 1pm-5;30 <br>
. Take over the tank B and C arround 1pm-5;30 <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
<br><b>Aug 19</b> <br>
<br><b>Aug 19</b> <br>
Line 2,145: Line 2,168:
. Inoue competent cells <br>
. Inoue competent cells <br>
. pBAD-key3-dTT restriction check <br>
. pBAD-key3-dTT restriction check <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
<br><b>Aug 20</b> <br>
<br><b>Aug 20</b> <br>
Line 2,154: Line 2,183:
. PCR check for J23102, LOCK 3 GFP dtt <br>
. PCR check for J23102, LOCK 3 GFP dtt <br>
. restrcition check for key1 dtt <br>
. restrcition check for key1 dtt <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
<br><b>Aug 21</b> <br>
<br><b>Aug 21</b> <br>
Line 2,167: Line 2,202:
. transform HKUSTlock.GFP.dTT <br>
. transform HKUSTlock.GFP.dTT <br>
. Transform pSB1C3-HKUSTlock <br>
. Transform pSB1C3-HKUSTlock <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
<br><b>Aug 22</b> <br>
<br><b>Aug 22</b> <br>
Line 2,183: Line 2,226:
. inoculate HKUSTlock1.GFP.dTT <br>
. inoculate HKUSTlock1.GFP.dTT <br>
. inoculate pSB1C3-HKUSTlock1 <br>
. inoculate pSB1C3-HKUSTlock1 <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
<br><b>Aug 23</b> <br>
<br><b>Aug 23</b> <br>
Line 2,190: Line 2,239:
. transform ust key <br>
. transform ust key <br>
. Check competent cells <br>
. Check competent cells <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
<br><b>Aug 24</b> <br>
<br><b>Aug 24</b> <br>
Line 2,195: Line 2,251:
. Colony PCR for Pbad.Key1.dTT <br>
. Colony PCR for Pbad.Key1.dTT <br>
. Gel check for Pbad.Key1.dTT <br>
. Gel check for Pbad.Key1.dTT <br>
-
 
+
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
Line 2,213: Line 2,274:
. transform ust key <br>
. transform ust key <br>
. ligation again key 1 into B0015 <br>
. ligation again key 1 into B0015 <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
<br><b>Aug 26</b> <br>
<br><b>Aug 26</b> <br>
Line 2,229: Line 2,297:
. single cut check for TAs <br>
. single cut check for TAs <br>
. make 5 alpha cell <br>
. make 5 alpha cell <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
<br><b>Aug 27</b> <br>
<br><b>Aug 27</b> <br>
Line 2,249: Line 2,325:
. overnight digestion of B0034 for control construct <br>
. overnight digestion of B0034 for control construct <br>
. Digestion of HKUST key-B0015 <br>
. Digestion of HKUST key-B0015 <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
<br><b>Aug 28</b> <br>
<br><b>Aug 28</b> <br>
Line 2,276: Line 2,361:
. parallel ligation and aneling for key 1 <br>
. parallel ligation and aneling for key 1 <br>
. Sequencing preparations <br>
. Sequencing preparations <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
<br><b>Aug 29</b> <br>
<br><b>Aug 29</b> <br>
Line 2,287: Line 2,379:
. Glycerol stock for locks <br>
. Glycerol stock for locks <br>
. PCR check for biobrick key1 <br>
. PCR check for biobrick key1 <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
<br><b>Aug 30</b> <br>
<br><b>Aug 30</b> <br>
Line 2,296: Line 2,395:
. Competent cells <br>
. Competent cells <br>
. AseI check Key 1 Dtt <br>
. AseI check Key 1 Dtt <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
       </div>
       </div>
Line 2,332: Line 2,438:
. inoculate orthogonality candidates <br>
. inoculate orthogonality candidates <br>
. Prepara midi-prep for 3k3 <br>
. Prepara midi-prep for 3k3 <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,347: Line 2,459:
. digestion of B0034-GFP-dtt <br>
. digestion of B0034-GFP-dtt <br>
. digestion of sherlock-GFP-dtt <br>
. digestion of sherlock-GFP-dtt <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
Line 2,375: Line 2,493:
. transform lock3c RBS.GFP.dTT <br>
. transform lock3c RBS.GFP.dTT <br>
. Midi prep preparations <br>
. Midi prep preparations <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,393: Line 2,518:
. Midi-prep preparations <br>
. Midi-prep preparations <br>
. Gel purification <br>
. Gel purification <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
Line 2,400: Line 2,531:
. Transform Pbad dTT <br>
. Transform Pbad dTT <br>
. Transform ligated produce of J23102 MCR GFP dTT with Pbad dTT <br>
. Transform ligated produce of J23102 MCR GFP dTT with Pbad dTT <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,414: Line 2,551:
·      <br>
·      <br>
·   <br>
·   <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,422: Line 2,563:
·      <br>
·      <br>
·   <br>
·   <br>
 +
·      <br>
 +
·      <br>
<br><b>Sept 10</b> <br>
<br><b>Sept 10</b> <br>
Line 2,432: Line 2,575:
. gel check for colony pcr <br>
. gel check for colony pcr <br>
. colony pcr <br>
. colony pcr <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,456: Line 2,605:
. Digestion of CLON 036 <br>
. Digestion of CLON 036 <br>
. colony pcr mcr mta <br>
. colony pcr mcr mta <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
       </div>
       </div>
Line 2,481: Line 2,636:
. plasmid PCR B1, B2 and B3 using sense oligo and VR <br>
. plasmid PCR B1, B2 and B3 using sense oligo and VR <br>
. Overnight digestion for ones which give positive results <br>
. Overnight digestion for ones which give positive results <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,490: Line 2,652:
. 2xYT (agar) AMP <br>
. 2xYT (agar) AMP <br>
. transform all <br>
. transform all <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,504: Line 2,672:
. Check "Diagonal constructs" and controls <br>
. Check "Diagonal constructs" and controls <br>
. migrate CR crontruts into 3K3 <br>
. migrate CR crontruts into 3K3 <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,512: Line 2,687:
. restrick check sherlock <br>
. restrick check sherlock <br>
. extrations.... madness <br>
. extrations.... madness <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
<br><b>Sept 20</b> <br>
<br><b>Sept 20</b> <br>
Line 2,518: Line 2,700:
. Colony PCR for CR.TA <br>
. Colony PCR for CR.TA <br>
. Colony PCR for CR.TA orthogonality <br>
. Colony PCR for CR.TA orthogonality <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
<br><b>Sept 21</b> <br>
<br><b>Sept 21</b> <br>
Line 2,523: Line 2,711:
. miniprep martching CR.TA <br>
. miniprep martching CR.TA <br>
. Restriction check by EcoR1 and Pst1 <br>
. Restriction check by EcoR1 and Pst1 <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,540: Line 2,735:
. transformation <br>
. transformation <br>
. pSB3K3- CRs ligation <br>
. pSB3K3- CRs ligation <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,556: Line 2,758:
. inoculation for HKUST lock Construct <br>
. inoculation for HKUST lock Construct <br>
. colony PCR For lock <br>
. colony PCR For lock <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
Line 2,565: Line 2,773:
. miniprep for biobrick submission <br>
. miniprep for biobrick submission <br>
. send plamsid for sequencing <br>
. send plamsid for sequencing <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
Line 2,572: Line 2,787:
. restriction check <br>
. restriction check <br>
. Gel check for previous PvuII digestions <br>
. Gel check for previous PvuII digestions <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,586: Line 2,808:
. Restriction check for controls for MCR/MTA <br>
. Restriction check for controls for MCR/MTA <br>
. Transformation of confirmed plasmid <br>
. Transformation of confirmed plasmid <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
<br><b>Sept 27</b> <br>
<br><b>Sept 27</b> <br>
Line 2,597: Line 2,824:
. transformed H6 H7 <br>
. transformed H6 H7 <br>
. O/N innoculation of MCR/MTA for characterization <br>
. O/N innoculation of MCR/MTA for characterization <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
<br><b>Sept 28</b> <br>
<br><b>Sept 28</b> <br>
Line 2,611: Line 2,844:
. Fluorescence measurement of MCR/MTA for characterization. <br>
. Fluorescence measurement of MCR/MTA for characterization. <br>
. Pick a colony dilute and spread MCR/MTA (we don't want bacteria lawn) <br>
. Pick a colony dilute and spread MCR/MTA (we don't want bacteria lawn) <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
Line 2,632: Line 2,871:
. Fluorescence measurement of MCR/MTA for characterization (second trial) <br>
. Fluorescence measurement of MCR/MTA for characterization (second trial) <br>
. Innoculate Lock and Keys for crude functional assay. <br>
. Innoculate Lock and Keys for crude functional assay. <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
Line 2,644: Line 2,888:
. ligation of H series and some from other series... <br>
. ligation of H series and some from other series... <br>
. transformtation of 16 confirmed constructs <br>
. transformtation of 16 confirmed constructs <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
<br><b>Oct 1</b> <br>
<br><b>Oct 1</b> <br>
Line 2,649: Line 2,898:
. digestion of key3,HKUSTkey? <br>
. digestion of key3,HKUSTkey? <br>
. miniprep- final constructs <br>
. miniprep- final constructs <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>

Revision as of 16:39, 16 October 2014



Notebook

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.






Pneumosensor

June 2014

Week 1
Week 2
Week 3
Week 4
Week 5

July 2014

Week 1
Week 2
Week 3
Week 4
Week 5

August 2014

Week 1
Week 2
Week 3
Week 4
Week 5

September 2014

Week 1
Week 2
Week 3
Week 4
Week 5





Riboregulator

June 2014

Week 1
Week 2
Week 3
Week 4
Week 5

July 2014

Week 1
Week 2
Week 3
Week 4
Week 5

August 2014

Week 1
Week 2
Week 3
Week 4
Week 5

September 2014

Week 1
Week 2
Week 3
Week 4
Week 5


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