Team:Hong Kong HKUST/wetlab/notebook

From 2014.igem.org

(Difference between revisions)
Line 1,611: Line 1,611:
. Primer Design for TA construct Gibson Assembly <br>
. Primer Design for TA construct Gibson Assembly <br>
. Gel check for the colony PCR <br>
. Gel check for the colony PCR <br>
-
. Pbad PCR amplification <br>
+
. P<sub>bad</sub> PCR amplification <br>
Line 1,621: Line 1,621:
. Colony PCR for CRs and TAs <br>
. Colony PCR for CRs and TAs <br>
. Re-ligation of TAs and CRs <br>
. Re-ligation of TAs and CRs <br>
-
. Pbad PCR clean up and Digestion <br>
+
. P<sub>bad</sub> PCR clean up and Digestion <br>
-
. Ligation of Pbad with pSB1C3 <br>
+
. Ligation of P<sub>bad</sub> with pSB1C3 <br>
-
. Sequencing preparation of Pbad <br>
+
. Sequencing preparation of P<sub>bad</sub> <br>
Line 1,634: Line 1,634:
<br> <b> July 10</b> <br>
<br> <b> July 10</b> <br>
Wet lab<br>
Wet lab<br>
-
. Sequencing preparation of Pbad <br>
+
. Sequencing preparation of P<sub>bad</sub> <br>
. Miniprep previous TAs CRs constructs <br>
. Miniprep previous TAs CRs constructs <br>
. Restrict check previous TAs CRs constructs <br>
. Restrict check previous TAs CRs constructs <br>
Line 1,643: Line 1,643:
<br> <b> July 11</b> <br>
<br> <b> July 11</b> <br>
Wet lab<br>
Wet lab<br>
-
. Pbad digestion and gel purification, Digestion of PBAD-pSB1C3 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 pBAD and E0240 <br>
+
. Ligation of P<sub>bad</sub> and E0240 <br>
. Overlaping PCR design <br>
. Overlaping PCR design <br>
. Gibson assembly master mix preparation <br>
. Gibson assembly master mix preparation <br>
Line 1,665: Line 1,665:
. Gibson assembly master mix preparation <br>
. Gibson assembly master mix preparation <br>
. Trevor's PCR <br>
. Trevor's PCR <br>
-
. Ligation of pBAD an E0240 <br>
+
. Ligation of P<sub>bad</sub> an E0240 <br>
. Competent cells preparations for DH5a and DH10B <br>
. Competent cells preparations for DH5a and DH10B <br>
. Prepare receptor backbone for oligo anealing, pSB1C3-J04450 (EcoRI, SpeI) <br>
. Prepare receptor backbone for oligo anealing, pSB1C3-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>
-
. Preparations for Pbad re-Characterization <br>
+
. Preparations for P<sub>bad</sub> re-Characterization <br>
<br> <b> July 15 </b> <br>
<br> <b> July 15 </b> <br>
Wet lab<br>
Wet lab<br>
-
. Inoculation for pBAD-GFP-TT <br>
+
. Inoculation for P<sub>bad</sub> -GFP-TT <br>
. (2nd time) Colony PCR usning VF2 and VR <br>
. (2nd time) Colony PCR usning VF2 and VR <br>
-
. Design primer for pBAD promoter sequencing <br>
+
. Design primer for P<sub>bad</sub> promoter sequencing <br>
. Gibson assembly master mix preparation <br>
. Gibson assembly master mix preparation <br>
-
. Preparations for Pbad re-characterization <br>
+
. Preparations for P<sub>bad</sub>  re-characterization <br>
. Competent cells preparations for DH5a and DH10B <br>
. Competent cells preparations for DH5a and DH10B <br>
. One full set re-ligation (O/N) <br>
. One full set re-ligation (O/N) <br>
Line 1,688: Line 1,688:
Wet lab<br>
Wet lab<br>
. Gibson assembly master mix preparation <br>
. Gibson assembly master mix preparation <br>
-
. Preparations for Pbad re-characterization <br>
+
. Preparations for P<sub>bad</sub> re-characterization <br>
. Competent cells preparations for DH5a and DH10B <br>
. Competent cells preparations for DH5a and DH10B <br>
. Prepare receptor backbone for oligo anealing <br>
. Prepare receptor backbone for oligo anealing <br>
Line 1,697: Line 1,697:
Wet lab<br>
Wet lab<br>
. Gibson assembly master mix preparation <br>
. Gibson assembly master mix preparation <br>
-
. Pbad re-characterization <br>
+
. P<sub>bad</sub> re-characterization <br>
. Miniprep CRs TAs <br>
. Miniprep CRs TAs <br>
. Restric check CRs,TAs <br>
. Restric check CRs,TAs <br>
Line 1,715: Line 1,715:
Wet lab<br>
Wet lab<br>
. Gibson assembly for amilCP <br>
. Gibson assembly for amilCP <br>
-
. Pbad re-characterization <br>
+
. P<sub>bad</sub>  re-characterization <br>
. Colony PCR <br>
. Colony PCR <br>
. Prepare receptor backbone for oligo anealing <br>
. Prepare receptor backbone for oligo anealing <br>
Line 1,722: Line 1,722:
. M9 mininaml medium preparation <br>
. M9 mininaml medium preparation <br>
. Colony PCR <br>
. Colony PCR <br>
-
. plate streaking for pBAD-E0240 <br>
+
. plate streaking for P<sub>bad</sub>-E0240 <br>
. inoculation of TAs and CRs <br>
. inoculation of TAs and CRs <br>
<br> <b> July 19</b> <br>
<br> <b> July 19</b> <br>
Wet lab<br>
Wet lab<br>
-
. Pbad re-characterization <br>
+
. P<sub>bad</sub> re-characterization <br>
Line 1,742: Line 1,742:
. Restrict check pSB1C3-TAs/CRs constructs <br>
. Restrict check pSB1C3-TAs/CRs constructs <br>
. Restrict GFP.dTT for CRs <br>
. Restrict GFP.dTT for CRs <br>
-
. Restrict Pbad for TAs <br>
+
. Restrict P<sub>bad</sub> for TAs <br>
. Restrict B0015 EcoR1 and XbaI <br>
. Restrict B0015 EcoR1 and XbaI <br>
. J04450 backbone extraction <br>
. J04450 backbone extraction <br>
. Colony PCR <br>
. Colony PCR <br>
. inoculation of TAs and CRs <br>
. inoculation of TAs and CRs <br>
-
. prepare Pbad L(+) A. concentration gradients for charac <br>
+
. prepare P<sub>bad</sub>L(+) A. concentration gradients for charac <br>
. Restrict CRs <br>
. Restrict CRs <br>
. Restrict TAs-Version1 <br>
. Restrict TAs-Version1 <br>
Line 1,753: Line 1,753:
. Ligate CR.GFP.dTT <br>
. Ligate CR.GFP.dTT <br>
. Ligate TA.dTT <br>
. Ligate TA.dTT <br>
-
. Transform Pbad.TA <br>
+
. Transform P<sub>bad</sub>.TA <br>
. Transform CR.GFP.dTT <br>
. Transform CR.GFP.dTT <br>
. Transform Gibson assembly <br>
. Transform Gibson assembly <br>
Line 1,773: Line 1,773:
. Gibson PCR <br>
. Gibson PCR <br>
. Teach inoue competent cells to main project <br>
. Teach inoue competent cells to main project <br>
-
. Miniprep of B0015, pBAD-E0240 <br>
+
. Miniprep of B0015, P<sub>bad</sub>-E0240 <br>
-
. glycerol stock (pBAD-E0240) <br>
+
. glycerol stock (P<sub>bad</sub>-E0240) <br>
. Re-Re-ligation of oligo annealing O/N <br>
. Re-Re-ligation of oligo annealing O/N <br>
Line 1,804: Line 1,804:
. innoculate last ligation <br>
. innoculate last ligation <br>
. Check past locks/keys status <br>
. Check past locks/keys status <br>
-
. Pbad sequencing <br>
+
. P<sub>bad</sub> sequencing <br>
. transformation of some of the existing riboregulator biobricks <br>
. transformation of some of the existing riboregulator biobricks <br>
. Benchling Check <br>
. Benchling Check <br>
Line 1,822: Line 1,822:
. ligate oligos itno B0015 <br>
. ligate oligos itno B0015 <br>
. prepare M9 for characterization <br>
. prepare M9 for characterization <br>
-
. transform our pbad 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 pSB1C3 ( with a control) <br>
. transformation of Clon001-010 in pSB1C3 ( with a control) <br>
Line 1,833: Line 1,833:
. If construct after gel check inoculation, restriction, ligation <br>
. If construct after gel check inoculation, restriction, ligation <br>
. prepare I20260 in PSB1C3 <br>
. prepare I20260 in PSB1C3 <br>
-
. transform our pbad into DH5alpha <br>
+
. transform our P<sub>bad</sub>into DH5alpha <br>
. prepare M9 for characterization <br>
. prepare M9 for characterization <br>
. Teach inoue competent cells to main project <br>
. Teach inoue competent cells to main project <br>
Line 1,883: Line 1,883:
. ligate CLON 005 with B0015 <br>
. ligate CLON 005 with B0015 <br>
. prepare backbone : B0015 E, X /// I13401 E,X /// J23102 X, P <br>
. prepare backbone : B0015 E, X /// I13401 E,X /// J23102 X, P <br>
-
. Ligate PBad with Key1 dTT <br>
+
. Ligate P<sub>bad</sub> with Key1 dTT <br>
. digest ptet lock 1 with S and P <br>
. digest ptet lock 1 with S and P <br>
Line 1,901: Line 1,901:
. Restrict TAs <br>
. Restrict TAs <br>
. restrict CRs <br>
. restrict CRs <br>
-
. Restrict pBAD for TAs <br>
+
. Restrict P<sub>bad</sub> for TAs <br>
. Restrict GFP.dTT for CRs <br>
. Restrict GFP.dTT for CRs <br>
. Restrict B0015 <br>
. Restrict B0015 <br>
-
. Ligate Pbad.TA <br>
+
. Ligate P<sub>bad</sub>.TA <br>
. liagte CR.GFP.dTT <br>
. liagte CR.GFP.dTT <br>
-
. Ligate Pbad.dTT <br>
+
. Ligate P<sub>bad</sub>.dTT <br>
. Transform TAs, CRs <br>
. Transform TAs, CRs <br>
. inoculate 001-010 <br>
. inoculate 001-010 <br>
Line 1,912: Line 1,912:
. inoculate B0015 <br>
. inoculate B0015 <br>
. inoculate constitutive promoter <br>
. inoculate constitutive promoter <br>
-
. Innoculate Pbad with key 1 dTT <br>
+
. Innoculate P<sub>bad</sub> with key 1 dTT <br>
. Competent cells preparations DH10B <br>
. Competent cells preparations DH10B <br>
. Prepare for functional assay of Pbad promoter <br>
. Prepare for functional assay of Pbad promoter <br>
Line 2,157: Line 2,157:
. transform HKUSTlock.GFP.dTT <br>
. transform HKUSTlock.GFP.dTT <br>
. Transform pSB1C3-HKUSTlock <br>
. Transform pSB1C3-HKUSTlock <br>
-
·      <br>
+
 
<br><b>Aug 22</b> <br>
<br><b>Aug 22</b> <br>
Line 2,174: Line 2,174:
. inoculate HKUSTlock1.GFP.dTT <br>
. inoculate HKUSTlock1.GFP.dTT <br>
. inoculate pSB1C3-HKUSTlock1 <br>
. inoculate pSB1C3-HKUSTlock1 <br>
-
·      <br>
+
 
<br><b>Aug 23</b> <br>
<br><b>Aug 23</b> <br>
Line 2,182: Line 2,182:
. transform ust key <br>
. transform ust key <br>
. Check competent cells <br>
. Check competent cells <br>
-
·      <br>
+
 
Line 2,189: Line 2,189:
. 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>
+
 
Line 2,207: Line 2,207:
. transform ust key <br>
. transform ust key <br>
. ligation again key 1 into B0015 <br>
. ligation again key 1 into B0015 <br>
-
·      <br>
+
 
<br><b>Aug 26</b> <br>
<br><b>Aug 26</b> <br>
Line 2,224: Line 2,224:
. single cut check for TAs <br>
. single cut check for TAs <br>
. make 5 alpha cell <br>
. make 5 alpha cell <br>
-
·      <br>
+
 
Line 2,246: Line 2,246:
. 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>
+
 
Line 2,275: Line 2,275:
. parallel ligation and aneling for key 1 <br>
. parallel ligation and aneling for key 1 <br>
. Sequencing preparations <br>
. Sequencing preparations <br>
-
·      <br>
+
 
Line 2,288: Line 2,288:
. Glycerol stock for locks <br>
. Glycerol stock for locks <br>
. PCR check for biobrick key1 <br>
. PCR check for biobrick key1 <br>
-
·      <br>
+
 
Line 2,299: Line 2,299:
. Competent cells <br>
. Competent cells <br>
. AseI check Key 1 Dtt <br>
. AseI check Key 1 Dtt <br>
-
·      <br>
+
 
Line 2,337: Line 2,337:
. inoculate orthogonality candidates <br>
. inoculate orthogonality candidates <br>
. Prepara midi-prep for 3k3 <br>
. Prepara midi-prep for 3k3 <br>
-
·      <br>
+
 
Line 2,354: Line 2,354:
. 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>
+
 
Line 2,383: Line 2,383:
. transform lock3c RBS.GFP.dTT <br>
. transform lock3c RBS.GFP.dTT <br>
. Midi prep preparations <br>
. Midi prep preparations <br>
-
·      <br>
+
 
Line 2,403: Line 2,403:
. Midi-prep preparations <br>
. Midi-prep preparations <br>
. Gel purification <br>
. Gel purification <br>
-
·      <br>
+
 
Line 2,411: Line 2,411:
. 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>
+
 
Line 2,451: Line 2,451:
. gel check for colony pcr <br>
. gel check for colony pcr <br>
. colony pcr <br>
. colony pcr <br>
-
·      <br>
+
 
-
·      <br>
+
-
·      <br>
+
-
·      <br>
+
-
·      <br>
+
Line 2,481: Line 2,477:
. Digestion of CLON 036 <br>
. Digestion of CLON 036 <br>
. colony pcr mcr mta <br>
. colony pcr mcr mta <br>
-
·      <br>
+
 
Line 2,500: Line 2,496:
Wet lab<br>
Wet lab<br>
. mini prep Pbad.key1.dtt <br>
. mini prep Pbad.key1.dtt <br>
-
. restrict check pbad.key1.dtt <br>
+
. restrict check P<sub>bad</sub>.key1.dtt <br>
. gel check for O/N c.pcr <br>
. gel check for O/N c.pcr <br>
-
. extract pbad.key1 <br>
+
. extract P<sub>bad</sub>.key1 <br>
. inoculate every CR.TA construct <br>
. inoculate every CR.TA construct <br>
. ligate transform key 1 with locks <br>
. ligate transform key 1 with locks <br>
Line 2,508: Line 2,504:
. 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>
+
Line 2,515: Line 2,510:
<br><b>Sept 17</b> <br>
<br><b>Sept 17</b> <br>
Wet lab<br>
Wet lab<br>
-
. purify pbad.key1 <br>
+
. purify P<sub>bad</sub>.key1 <br>
-
. ligate pbad key1 dtt with locks <br>
+
. ligate P<sub>bad</sub>key1 dtt with locks <br>
-
. transform locks.Pbad.key1.dtt <br>
+
. transform locks.P<sub>bad</sub>.key1.dtt <br>
. 2xYT (agar) AMP <br>
. 2xYT (agar) AMP <br>
. transform all <br>
. transform all <br>
-
·      <br>
+
 
Line 2,536: Line 2,531:
. 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>
+
 
Line 2,546: Line 2,541:
. restrick check sherlock <br>
. restrick check sherlock <br>
. extrations.... madness <br>
. extrations.... madness <br>
-
·      <br>
+
 
Line 2,554: Line 2,549:
. 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>
+
 
Line 2,561: Line 2,556:
. 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>
+
 
Line 2,580: Line 2,575:
. transformation <br>
. transformation <br>
. pSB3K3- CRs ligation <br>
. pSB3K3- CRs ligation <br>
-
·      <br>
+
 
Line 2,598: Line 2,593:
. inoculation for HKUST lock Construct <br>
. inoculation for HKUST lock Construct <br>
. colony PCR For lock <br>
. colony PCR For lock <br>
-
·      <br>
+
 
Line 2,608: Line 2,603:
. miniprep for biobrick submission <br>
. miniprep for biobrick submission <br>
. send plamsid for sequencing <br>
. send plamsid for sequencing <br>
-
·      <br>
+
 
Line 2,616: Line 2,611:
. restriction check <br>
. restriction check <br>
. Gel check for previous PvuII digestions <br>
. Gel check for previous PvuII digestions <br>
-
·      <br>
+
 
Line 2,632: Line 2,627:
. 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><b>Sept 27</b> <br>
<br><b>Sept 27</b> <br>
Line 2,644: Line 2,639:
. 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>
+
 
Line 2,660: Line 2,655:
. 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>
+
 
Line 2,683: Line 2,678:
. 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>
+
 
Line 2,696: Line 2,691:
. 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><b>Oct 1</b> <br>
<br><b>Oct 1</b> <br>
Line 2,702: Line 2,697:
. digestion of key3,HKUSTkey? <br>
. digestion of key3,HKUSTkey? <br>
. miniprep- final constructs <br>
. miniprep- final constructs <br>
-
·      <br>
+
 

Revision as of 12:26, 17 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.






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Riboregulator

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