Team:Hong Kong HKUST/wetlab/notebook

From 2014.igem.org

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. ligation of TAs and CRs <br>
. ligation of TAs and CRs <br>
. Colony PCR <br>
. Colony PCR <br>
-
. Digest Receptor Backbone pSB3K3 <br>
+
. Digest Receptor Backbone pSB1C3 <br>
. Agar-LB plates <br>
. Agar-LB plates <br>
. Inoculation for transformed Biobricks <br>
. Inoculation for transformed Biobricks <br>
Line 1,345: Line 1,346:
. Re-ligation of TAs and CRs <br>
. Re-ligation of TAs and CRs <br>
. P<sub>bad</sub> PCR clean up and Digestion <br>
. P<sub>bad</sub> PCR clean up and Digestion <br>
-
. Ligation of P<sub>bad</sub> with pSB3K3 <br>
+
. Ligation of P<sub>bad</sub> with pSB1C3 <br>
. Sequencing preparation of P<sub>bad</sub> <br>
. Sequencing preparation of P<sub>bad</sub> <br>
Line 1,366: Line 1,367:
<br> <b> July 11</b> <br>
<br> <b> July 11</b> <br>
Wet lab<br>
Wet lab<br>
-
. P<sub>bad</sub> digestion and gel purification, Digestion of P<sub>bad</sub>-pSB3K3 with EcoR1-HF and Spe1 and gel purification <br>
+
. P<sub>bad</sub> digestion and gel purification, Digestion of P<sub>bad</sub>-pSB1C3 with EcoR1-HF and Spe1 and gel purification <br>
. Ligation of P<sub>bad</sub> and BBa_E0240 <br>
. Ligation of P<sub>bad</sub> and BBa_E0240 <br>
. Overlapping PCR design <br>
. Overlapping PCR design <br>
Line 1,382: Line 1,383:
   <div>
   <div>
-
       <a href="#" class="head">Week 3</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> July 14 </b> <br>
<br> <b> July 14 </b> <br>
Wet lab<br>
Wet lab<br>
. Gibson assembly master mix preparation <br>
. Gibson assembly master mix preparation <br>
-
. Trevor's PCR <br>
+
. PCR <br>
. Ligation of P<sub>bad</sub> an BBa_E0240 <br>
. Ligation of P<sub>bad</sub> an BBa_E0240 <br>
. Competent cells preparations for DH5alpha and DH10B <br>
. Competent cells preparations for DH5alpha and DH10B <br>
-
. Prepare receptor backbone for oligo anealing, pSB3K3-BBa_J04450 (EcoRI, SpeI) <br>
+
. Prepare receptor backbone for oligo anealing, pSB1C3-BBa_J04450 (EcoRI, SpeI) <br>
. One full set of re-ligation (short time) <br>
. One full set of re-ligation (short time) <br>
. One full set of re-ligation (O/N) <br>
. One full set of re-ligation (O/N) <br>
Line 1,457: Line 1,458:
   <div>
   <div>
-
       <a href="#" class="head">Week 4</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> July 21 </b> <br>
<br> <b> July 21 </b> <br>
Line 1,463: Line 1,464:
. Gibson assembly for amilCP <br>
. Gibson assembly for amilCP <br>
. Miniprep CRs TAs <br>
. Miniprep CRs TAs <br>
-
. restriction check pSB3K3-TAs/CRs constructs <br>
+
. restriction check pSB1C3-TAs/CRs constructs <br>
. Restrict GFP.dTT for CRs <br>
. Restrict GFP.dTT for CRs <br>
. Restrict P<sub>bad</sub> for TAs <br>
. Restrict P<sub>bad</sub> for TAs <br>
Line 1,485: Line 1,486:
. Colony PCR <br>
. Colony PCR <br>
. Miniprep CRs TAs <br>
. Miniprep CRs TAs <br>
-
. restriction check pSB3K3-TAs/CRs constructs <br>
+
. restriction check pSB1C3-TAs/CRs constructs <br>
. Restrict BBa_B0015-Version2 <br>
. Restrict BBa_B0015-Version2 <br>
Line 1,503: Line 1,504:
Wet lab<br>
Wet lab<br>
. miniprep candidate colony or construct <br>
. miniprep candidate colony or construct <br>
-
. restriction check pSB3K3-TAs/CRs constructs <br>
+
. restriction check pSB1C3-TAs/CRs constructs <br>
. Re-Re-ligation transformation <br>
. Re-Re-ligation transformation <br>
. Re-Re-ligation transformation V.X <br>
. Re-Re-ligation transformation V.X <br>
Line 1,516: Line 1,517:
   <div>
   <div>
-
       <a href="#" class="head">Week 5</a>
+
       <a href="#" class="head">Week 4</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
<br> <b> July 28 </b> <br>
<br> <b> July 28 </b> <br>
Line 1,535: Line 1,536:
Wet lab<br>
Wet lab<br>
. Miniprep and restriction check inoculations <br>
. Miniprep and restriction check inoculations <br>
-
. restriction check pSB3K3-TAs/CRs constructs <br>
+
. restriction check pSB1C3-TAs/CRs constructs <br>
. Inoculation of CR and TA from distribution kit <br>
. Inoculation of CR and TA from distribution kit <br>
-
. prepare BBa_I20260 in pSB3K3 <br>
+
. prepare BBa_I20260 in pSB1C3 <br>
. prepare Side project Buffer <br>
. prepare Side project Buffer <br>
. prepare side project gel red 200:1 <br>
. prepare side project gel red 200:1 <br>
Line 1,547: Line 1,548:
. transform our P<sub>bad</sub> into DH5alpha <br>
. transform our P<sub>bad</sub> into DH5alpha <br>
. prepare M9 medium <br>
. prepare M9 medium <br>
-
. transformation of Clon001-010 in pSB3K3 ( with a control) <br>
+
. transformation of Clon001-010 in pSB1C3 ( with a control) <br>
<br> <b> July 30</b> <br>
<br> <b> July 30</b> <br>
Wet lab<br>
Wet lab<br>
-
. transformation of CLON001-010 in pSB3K3 (BBa_B0015, BBa_I13401) <br>
+
. transformation of CLON001-010 in pSB1C3 (BBa_B0015, BBa_I13401) <br>
. C. PCR for yesterday transformations (CLON001-010) <br>
. C. PCR for yesterday transformations (CLON001-010) <br>
. If construct after gel check inoculation, restriction, ligation <br>
. If construct after gel check inoculation, restriction, ligation <br>
-
. prepare BBa_I20260 in pSB3K3 <br>
+
. prepare BBa_I20260 in pSB1C3 <br>
. transform our P<sub>bad</sub>into DH5alpha <br>
. transform our P<sub>bad</sub>into DH5alpha <br>
. prepare M9 for characterization <br>
. prepare M9 for characterization <br>
Line 1,564: Line 1,565:
. Try Gibson assembly mastermix <br>
. Try Gibson assembly mastermix <br>
. Digest Biobrick TA and CR <br>
. Digest Biobrick TA and CR <br>
-
. PCR clean up? for existing CR and TA Biobricks <br>
+
. PCR clean up for existing CR and TA Biobricks <br>
. run gel for yesterday's colony PCR <br>
. run gel for yesterday's colony PCR <br>
. check dNTPs <br>
. check dNTPs <br>
Line 1,570: Line 1,571:
. colony PCR for 5th <br>
. colony PCR for 5th <br>
. inoculate candidates <br>
. inoculate candidates <br>
-
. prepare BBa_I20260 in pSB3K3 <br>
+
. prepare BBa_I20260 in pSB1C3 <br>
Line 1,664: Line 1,665:
. Glycerol stock for 001-010 <br>
. Glycerol stock for 001-010 <br>
. Inoue competent cells <br>
. Inoue competent cells <br>
-
. "Mini Prep pSB3K3-PCRP<sub>bad</sub><br>
+
. "Mini Prep pSB1C3-PCR P<sub>bad</sub><br>
<br><b>Aug 6</b> <br>
<br><b>Aug 6</b> <br>
Line 1,839: Line 1,840:
. Inoculate candidates <br>
. Inoculate candidates <br>
. Prepare pSB3K3 <br>
. Prepare pSB3K3 <br>
-
. Prepare pSB3K3 for oligo ligation <br>
+
. Prepare pSB1C3 for oligo ligation <br>
. Prepare BBa_I13401 for oligo ligation <br>
. Prepare BBa_I13401 for oligo ligation <br>
. Prepare BBa_B0015 for oligo ligation <br>
. Prepare BBa_B0015 for oligo ligation <br>
Line 1,853: Line 1,854:
. miniprep possible candidates <br>
. miniprep possible candidates <br>
. Restrict TA.dTT <br>
. Restrict TA.dTT <br>
-
. pSB3K3-BBa_I13401 for oligo ligation <br>
+
. pSB1C3-BBa_I13401 for oligo ligation <br>
. BBa_B0015 for oligo ligation <br>
. BBa_B0015 for oligo ligation <br>
-
. pSB3K3-BBa_J04450 for oligo ligation <br>
+
. pSB1C3-BBa_J04450 for oligo ligation <br>
. Inoue competent cells <br>
. Inoue competent cells <br>
. P<sub>bad</sub>-key3-dTT restriction check <br>
. P<sub>bad</sub>-key3-dTT restriction check <br>
Line 1,863: Line 1,864:
. Gel check for P<sub>bad</sub>.TA.dTTs <br>
. Gel check for P<sub>bad</sub>.TA.dTTs <br>
. purification for BBa_B0015, BBa_I13401 <br>
. purification for BBa_B0015, BBa_I13401 <br>
-
. pSB3K3-BBa_J04450 for oligo ligation <br>
+
. pSB1C3-BBa_J04450 for oligo ligation <br>
. Gel check for PCR products (19/08) <br>
. Gel check for PCR products (19/08) <br>
. PCR check for BBa_J23102, LOCK 3 GFP DTT <br>
. PCR check for BBa_J23102, LOCK 3 GFP DTT <br>
Line 1,879: Line 1,880:
. Glycerol stocks <br>
. Glycerol stocks <br>
. transform HKUSTlock.GFP.dTT <br>
. transform HKUSTlock.GFP.dTT <br>
-
. Transform pSB3K3-HKUSTlock <br>
+
. Transform pSB1C3-HKUSTlock <br>
Line 1,885: Line 1,886:
Wet lab<br>
Wet lab<br>
. competent cells <br>
. competent cells <br>
-
. Colony PCR for pSB3K3-HKUSTlock1 <br>
+
. Colony PCR for pSB1C3-HKUSTlock1 <br>
. Colony PCR for HKUSTlock1.GFP.dTT <br>
. Colony PCR for HKUSTlock1.GFP.dTT <br>
. miniprep pSB3K3-J04550 for pSB3K3 Backbone <br>
. miniprep pSB3K3-J04550 for pSB3K3 Backbone <br>
Line 1,896: Line 1,897:
. extraction of pSB3K3 <br>
. extraction of pSB3K3 <br>
. inoculate HKUSTlock1.GFP.dTT <br>
. inoculate HKUSTlock1.GFP.dTT <br>
-
. inoculate pSB3K3-HKUSTlock1 <br>
+
. inoculate pSB1C3-HKUSTlock1 <br>
Line 1,902: Line 1,903:
Wet lab<br>
Wet lab<br>
. Gel check for TAs <br>
. Gel check for TAs <br>
-
. colony PCR for UST lock in BBa_I13401 and pSB3K3 <br>
+
. colony PCR for UST lock in BBa_I13401 and pSB1C3 <br>
. transform ust key <br>
. transform ust key <br>
. Check competent cells <br>
. Check competent cells <br>
Line 1,942: Line 1,943:
. PCR check for CR.GFP.dTTs <br>
. PCR check for CR.GFP.dTTs <br>
. Inoculate all possible candidates <br>
. Inoculate all possible candidates <br>
-
. Feeling paranoic? Prepare BBa_I13401 for CRs <br>
+
. Prepare BBa_I13401 for CRs <br>
. Streak possible c.p.CR/RBS.GFP.dTT <br>
. Streak possible c.p.CR/RBS.GFP.dTT <br>
. make SOC <br>
. make SOC <br>
. single cut check for TAs <br>
. single cut check for TAs <br>
-
. make DH5alpha cell <br>
+
. make 5 alpha cell <br>
Line 1,956: Line 1,957:
. gel check for o/n PCR <br>
. gel check for o/n PCR <br>
. restriction check for ust key 1 <br>
. restriction check for ust key 1 <br>
-
. restrick check for key 1 <br>
+
. restriction check for key 1 <br>
. P<sub>bad</sub> for ust key 1 <br>
. P<sub>bad</sub> for ust key 1 <br>
. extract ust lock 1 for BBa_J23102 <br>
. extract ust lock 1 for BBa_J23102 <br>
Line 1,962: Line 1,963:
. transform ust lock and key <br>
. transform ust lock and key <br>
. prepare plates for macroscope <br>
. prepare plates for macroscope <br>
-
. prepare DNA for sequencing <br>
+
. prepare dna for sequencing <br>
. SOB <br>
. SOB <br>
. SOC <br>
. SOC <br>
Line 1,987: Line 1,988:
. Key one restriction check <br>
. Key one restriction check <br>
. key one restriction for inserting P<sub>bad</sub> <br>
. key one restriction for inserting P<sub>bad</sub> <br>
-
. Restrict and exytact lock3c rbs and ligate into c.promoter <br>
+
. Restrict and extract lock3c rbs and ligate into c.promoter <br>
. ligate lock 3c.GFP.dTT with C. promoter <br>
. ligate lock 3c.GFP.dTT with C. promoter <br>
-
. transform c. promoterlock 3c rbs.GFP.DTT <br>
+
. transform c. promoter lock 3c rbs.GFP.DTT <br>
. Extract mcr/mta <br>
. Extract mcr/mta <br>
. ligate MCR and MTA into pSB3K3 <br>
. ligate MCR and MTA into pSB3K3 <br>
Line 1,996: Line 1,997:
. BBa_E0240 ligate with C. Promoter <br>
. BBa_E0240 ligate with C. Promoter <br>
. TB <br>
. TB <br>
-
. parallel ligation and aneling for key 1 <br>
+
. parallel ligation and anealing for key 1 <br>
. Sequencing preparations <br>
. Sequencing preparations <br>
Line 2,010: Line 2,011:
. transform MCR/MTA <br>
. transform MCR/MTA <br>
. Glycerol stock for locks <br>
. Glycerol stock for locks <br>
-
. PCR check for biobrick key1 <br>
+
. PCR check for Biobrick key1 <br>
Line 2,056: Line 2,057:
. Colony PCR for CR.TAs (orthogonality) <br>
. Colony PCR for CR.TAs (orthogonality) <br>
. Colony PCR for sherLock-GFP-DTT <br>
. Colony PCR for sherLock-GFP-DTT <br>
-
. restriction check for Lost Your Key in pSB3K3 <br>
+
. restriction check for Lost Your Key in pSB1C3 <br>
. transform P<sub>bad</sub>.lost your key.DTT <br>
. transform P<sub>bad</sub>.lost your key.DTT <br>
. inoculate orthogonality candidates <br>
. inoculate orthogonality candidates <br>
Line 2,074: Line 2,075:
. inoculation of B0034-GFP-DTT <br>
. inoculation of B0034-GFP-DTT <br>
. inoculation of Sherlock-GFP-DTT <br>
. inoculation of Sherlock-GFP-DTT <br>
-
. sequencing for sherlock in pSB3K3 <br>
+
. sequencing for sherlock in pSB1C3 <br>
. digestion of B0034-GFP-DTT <br>
. digestion of B0034-GFP-DTT <br>
. digestion of sherlock-GFP-DTT <br>
. digestion of sherlock-GFP-DTT <br>
Line 2,086: Line 2,087:
. digestion of P<sub>bad</sub> dTT <br>
. digestion of P<sub>bad</sub> dTT <br>
. digestion of BBa_J23102 MCR GFP dTT <br>
. digestion of BBa_J23102 MCR GFP dTT <br>
-
. digestion of BBa_J23102 BBa_E0240(B0032 GFP dTT) <br>
+
. digestion of BBa_J23102 BBa_E0240(BBa_B0032 GFP dTT) <br>
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450) <br>
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450) <br>
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
Line 2,101: Line 2,102:
. ligation of key1 DTT with P<sub>bad</sub> <br>
. ligation of key1 DTT with P<sub>bad</sub> <br>
. Gel check for O/N colonyPCR <br>
. Gel check for O/N colonyPCR <br>
-
. CRAZY PCR check <br>
+
. PCR check <br>
. Check 031 <br>
. Check 031 <br>
. ligate lock3c RBS.GFP.dTT into c.p plasmid <br>
. ligate lock3c RBS.GFP.dTT into c.p plasmid <br>
Line 2,115: Line 2,116:
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br>
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br>
-
. digestion of BBa_J23102 BBa_E0240(B0032 GFP dTT) <br>
+
. digestion of BBa_J23102 BBa_E0240(BBa_B0032 GFP dTT) <br>
. Transformation of ligated projects mentioned above <br>
. Transformation of ligated projects mentioned above <br>
. Colony PCR for sherLock-GFP-DTT <br>
. Colony PCR for sherLock-GFP-DTT <br>
Line 2,121: Line 2,122:
. Inoculation of successful colonies <br>
. Inoculation of successful colonies <br>
. Gel check for O/N Colony PCR <br>
. Gel check for O/N Colony PCR <br>
-
. Gel check for O/N crazy PCR <br>
+
. Gel check for O/N PCR <br>
. Gel check for Erics restriction check <br>
. Gel check for Erics restriction check <br>
. Inoculation of successful colonies <br>
. Inoculation of successful colonies <br>
Line 2,131: Line 2,132:
<br><b>Sept 5</b> <br>
<br><b>Sept 5</b> <br>
Wet lab<br>
Wet lab<br>
-
. miniprep/glycerol stock <br>
+
. miniprep/ glycerol stock <br>
. Transform P<sub>bad</sub> dTT <br>
. Transform P<sub>bad</sub> dTT <br>
. Transform ligated produce of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
. Transform ligated produce of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
Line 2,263: Line 2,264:
. ligate problematic cr.key <br>
. ligate problematic cr.key <br>
. restriction check sherlock <br>
. restriction check sherlock <br>
-
. extractions.... madness <br>
+
. gel extraction<br>
Line 2,294: Line 2,295:
. transform problematic sets <br>
. transform problematic sets <br>
. prepare DNA for submission <br>
. prepare DNA for submission <br>
-
. Gel purification for Doyle <br>
+
. Gel purification <br>
. Ligation lock-DTT with constitutive promoter <br>
. Ligation lock-DTT with constitutive promoter <br>
. transformation <br>
. transformation <br>
Line 2,310: Line 2,311:
. Agar L (+) plates <br>
. Agar L (+) plates <br>
. Send DNA to BGI for sequencing <br>
. Send DNA to BGI for sequencing <br>
-
. C. PCR? <br>
+
. C. PCR <br>
. Inoculate possible candidates <br>
. Inoculate possible candidates <br>
. ligate problematic sets <br>
. ligate problematic sets <br>
Line 2,321: Line 2,322:
<br><b>Sept 24</b> <br>
<br><b>Sept 24</b> <br>
Wet lab<br>
Wet lab<br>
-
. Competent cells preparations! <br>
+
. Competent cells preparations <br>
. minipreps <br>
. minipreps <br>
. colony PCR <br>
. colony PCR <br>
-
. miniprep for biobrick submission <br>
+
. miniprep for Biobrick submission <br>
. send plasmid for sequencing <br>
. send plasmid for sequencing <br>
Line 2,342: Line 2,343:
. Gel check PvuII digestions <br>
. Gel check PvuII digestions <br>
. Gel check AseI digestions <br>
. Gel check AseI digestions <br>
-
. 3A assembly for non pSB3K3 <br>
+
. 3A assembly for non pSB1C3 <br>
. final batch of ligation for all missing component <br>
. final batch of ligation for all missing component <br>
. prepare DNA for submission <br>
. prepare DNA for submission <br>
Line 2,367: Line 2,368:
<br><b>Sept 28</b> <br>
<br><b>Sept 28</b> <br>
Wet lab<br>
Wet lab<br>
-
. Extract pSB3K3 <br>
+
. Extract pSB1C3 <br>
. Extract 036 <br>
. Extract 036 <br>
. miniprep "H" series candidates <br>
. miniprep "H" series candidates <br>
Line 2,392: Line 2,393:
. restriction check AseI <br>
. restriction check AseI <br>
. restriction check NcoI <br>
. restriction check NcoI <br>
-
. mini prep 6 + MCR/MTA (thanks from doyle) <br>
+
. mini prep 6 + MCR/MTA (thanks from Doyle) <br>
. Restriction check for MCR/MTA in pSB3K3. <br>
. Restriction check for MCR/MTA in pSB3K3. <br>
. restrict 036 <br>
. restrict 036 <br>
Line 2,398: Line 2,399:
. ligate monkey with sherlock <br>
. ligate monkey with sherlock <br>
. Gel check for NcoI and AseI <br>
. Gel check for NcoI and AseI <br>
-
. Get pSB3K3 backbone EcoRI and PstI <br>
+
. Get pSB1C3 backbone EcoRI and PstI <br>
. Fluorescence measurement of MCR/MTA for characterization (second trial) <br>
. Fluorescence measurement of MCR/MTA for characterization (second trial) <br>
. inoculate Lock and Keys for crude functional assay. <br>
. inoculate Lock and Keys for crude functional assay. <br>
Line 2,409: Line 2,410:
. miniprep 11 tubes in shaker c <br>
. miniprep 11 tubes in shaker c <br>
. restriction check H" series with NcoI <br>
. restriction check H" series with NcoI <br>
-
. Restrict inoculated colonies with AseI <br>
+
. Restrict inoculated colonies wiht AseI <br>
-
. Restrict inoculated colonies with NcoI <br>
+
. Restrict inoculated colonies wiht NcoI <br>
. Gel purifications <br>
. Gel purifications <br>
. ligation of H series and some from other series... <br>
. ligation of H series and some from other series... <br>
Line 2,418: Line 2,419:
<br><b>Oct 1</b> <br>
<br><b>Oct 1</b> <br>
Wet lab<br>
Wet lab<br>
-
. digestion of key3,HKUSTkey? <br>
+
. digestion of key3,HKUSTkey<br>
-
. miniprep final constructs <br>
+
. miniprep- final constructs <br>
Line 2,453: Line 2,454:
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Latest revision as of 00:38, 18 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 Project Pneuumonsensor and Project Riboregulator are displayed separately.






Pneumosensor

June 2014

Week 1
Week 2
Week 3
Week 4

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

July 2014

Week 1
Week 2
Week 3
Week 4

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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Home

Pneumosensor

Riboregulator

Human Practice

Team

WetLab

Achievement