Team:Hong Kong HKUST/wetlab/notebook

From 2014.igem.org

(Difference between revisions)
Line 1,326: Line 1,326:
<br> <b> July 7 </b> <br>
<br> <b> July 7 </b> <br>
Wet lab<br>
Wet lab<br>
-
. Oligos phosphorilation <br>
+
. Oligos phosphorylation <br>
. ligation of TAs and CRs <br>
. ligation of TAs and CRs <br>
. Colony PCR <br>
. Colony PCR <br>
-
. Digest Receptor Backbone pSB1C3 <br>
+
. Digest Receptor Backbone pSB3K3 <br>
. Agar-LB plates <br>
. Agar-LB plates <br>
-
. Inoculation for transformded BioBricks <br>
+
. Inoculation for transformed Biobricks <br>
. 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>
Line 1,341: Line 1,341:
. Transformation of ligated TAs and CRs <br>
. Transformation of ligated TAs and CRs <br>
. Miniprep previous TAs CRs constructs <br>
. Miniprep previous TAs CRs constructs <br>
-
. Restrict check previous TAs CRs constructs <br>
+
. restriction check previous TAs CRs constructs <br>
. 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>
. 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 pSB1C3 <br>
+
. Ligation of P<sub>bad</sub> with pSB3K3 <br>
. Sequencing preparation of P<sub>bad</sub> <br>
. Sequencing preparation of P<sub>bad</sub> <br>
Line 1,359: Line 1,359:
. Sequencing preparation of P<sub>bad</sub> <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>
+
. restriction check previous TAs CRs constructs <br>
. Colony PCR <br>
. Colony PCR <br>
. Inoculation for glycerol stocks <br>
. Inoculation for glycerol stocks <br>
-
. Digest Receptor Backbone , digestion of BBa_ E0240 with EcoR1-HF and Xbal and gel purification <br>
+
. Digest Receptor Backbone , digestion of BBa_ BBa_E0240 with EcoR1-HF and Xbal and gel purification <br>
<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>-pSB1C3 with EcoR1-HF and Spe1 and gel purification <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>
-
. Ligation of P<sub>bad</sub> and E0240 <br>
+
. Ligation of P<sub>bad</sub> and BBa_E0240 <br>
-
. Overlaping PCR design <br>
+
. Overlapping PCR design <br>
. Gibson assembly master mix preparation <br>
. Gibson assembly master mix preparation <br>
. Glycerol stock <br>
. Glycerol stock <br>
-
. Competent cells preparations for DH5a and DH10B <br>
+
. Competent cells preparations for DH5alpha and DH10B <br>
. Run gel for yesterday's restriction check <br>
. Run gel for yesterday's restriction check <br>
-
. Miniprep E0240, mRFP, B0015 <br>
+
. Miniprep BBa_E0240, mRFP, BBa_B0015 <br>
. Colony PCR <br>
. Colony PCR <br>
. One full set re-ligation (short time) <br>
. One full set re-ligation (short time) <br>
-
. Miniprep I13401 and K592009 <br>
+
. Miniprep BBa_I13401 and BBa_K592009 <br>
       </div>
       </div>
Line 1,382: Line 1,382:
   <div>
   <div>
-
       <a href="#" class="head">Week 2</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 14 </b> <br>
<br> <b> July 14 </b> <br>
Line 1,388: Line 1,388:
. Gibson assembly master mix preparation <br>
. Gibson assembly master mix preparation <br>
. Trevor's PCR <br>
. Trevor's PCR <br>
-
. Ligation of P<sub>bad</sub> an E0240 <br>
+
. Ligation of P<sub>bad</sub> an BBa_E0240 <br>
-
. Competent cells preparations for DH5a and DH10B <br>
+
. Competent cells preparations for DH5alpha and DH10B <br>
-
. Prepare receptor backbone for oligo anealing, pSB1C3-J04450 (EcoRI, SpeI) <br>
+
. Prepare receptor backbone for oligo anealing, pSB3K3-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 1,399: Line 1,399:
Wet lab<br>
Wet lab<br>
. Inoculation for P<sub>bad</sub> -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 using VF2 and VR <br>
. Design primer for P<sub>bad</sub> 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 P<sub>bad</sub>  re-characterization <br>
. Preparations for P<sub>bad</sub>  re-characterization <br>
-
. Competent cells preparations for DH5a and DH10B <br>
+
. Competent cells preparations for DH5alpha and DH10B <br>
. One full set re-ligation (O/N) <br>
. One full set re-ligation (O/N) <br>
. Prepare receptor backbone for oligo anealing <br>
. Prepare receptor backbone for oligo anealing <br>
Line 1,412: Line 1,412:
. Gibson assembly master mix preparation <br>
. Gibson assembly master mix preparation <br>
. Preparations for P<sub>bad</sub> re-characterization <br>
. Preparations for P<sub>bad</sub> re-characterization <br>
-
. Competent cells preparations for DH5a and DH10B <br>
+
. Competent cells preparations for DH5alpha and DH10B <br>
. Prepare receptor backbone for oligo anealing <br>
. Prepare receptor backbone for oligo anealing <br>
-
. Innoculate succesful oligo annealing colonies <br>
+
. inoculate successful oligo annealing colonies <br>
. Oligo annealing products C. PCR? <br>
. Oligo annealing products C. PCR? <br>
Line 1,422: Line 1,422:
. P<sub>bad</sub> re-characterization <br>
. P<sub>bad</sub> re-characterization <br>
. Miniprep CRs TAs <br>
. Miniprep CRs TAs <br>
-
. Restric check CRs,TAs <br>
+
. Restriction check CRs,TAs <br>
. Oligo annealing products C. PCR <br>
. Oligo annealing products C. PCR <br>
. Prepare receptor backbone for oligo anealing <br>
. Prepare receptor backbone for oligo anealing <br>
Line 1,445: Line 1,445:
. M9 mininaml medium preparation <br>
. M9 mininaml medium preparation <br>
. Colony PCR <br>
. Colony PCR <br>
-
. plate streaking for P<sub>bad</sub>-E0240 <br>
+
. plate streaking for P<sub>bad</sub>-BBa_E0240 <br>
. inoculation of TAs and CRs <br>
. inoculation of TAs and CRs <br>
Line 1,457: Line 1,457:
   <div>
   <div>
-
       <a href="#" class="head">Week 3</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 21 </b> <br>
<br> <b> July 21 </b> <br>
Line 1,463: Line 1,463:
. Gibson assembly for amilCP <br>
. Gibson assembly for amilCP <br>
. Miniprep CRs TAs <br>
. Miniprep CRs TAs <br>
-
. Restrict check pSB1C3-TAs/CRs constructs <br>
+
. restriction check pSB3K3-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>
-
. Restrict B0015 EcoR1 and XbaI <br>
+
. Restrict BBa_B0015 EcoR1 and XbaI <br>
-
. J04450 backbone extraction <br>
+
. BBa_J04450 backbone extraction <br>
. Colony PCR <br>
. Colony PCR <br>
. inoculation of TAs and CRs <br>
. inoculation of TAs and CRs <br>
-
. prepare P<sub>bad</sub>L(+) A. concentration gradients for charac <br>
+
. prepare P<sub>bad</sub> L(+) A. concentration gradients for characterization <br>
. Restrict CRs <br>
. Restrict CRs <br>
. Restrict TAs-Version1 <br>
. Restrict TAs-Version1 <br>
Line 1,485: Line 1,485:
. Colony PCR <br>
. Colony PCR <br>
. Miniprep CRs TAs <br>
. Miniprep CRs TAs <br>
-
. Restrict check pSB1C3-TAs/CRs constructs <br>
+
. restriction check pSB3K3-TAs/CRs constructs <br>
-
. Restrict B0015-Version2 <br>
+
. Restrict BBa_B0015-Version2 <br>
Line 1,492: Line 1,492:
Wet lab<br>
Wet lab<br>
. Inoculation candidate colony or construct <br>
. Inoculation candidate colony or construct <br>
-
. Miniprep and restrict check innoculations <br>
+
. Miniprep and restriction check inoculations <br>
. Gibson assembly check <br>
. Gibson assembly check <br>
. 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, P<sub>bad</sub>-E0240 <br>
+
. Miniprep of BBa_B0015, P<sub>bad</sub>-BBa_E0240 <br>
-
. glycerol stock (P<sub>bad</sub>-E0240) <br>
+
. glycerol stock (P<sub>bad</sub>-BBa_E0240) <br>
-
. Re-Re-ligation of oligo annealing O/N <br>
+
. Re-ligation of oligo annealing O/N <br>
<br> <b> July 24</b> <br>
<br> <b> July 24</b> <br>
Wet lab<br>
Wet lab<br>
. miniprep candidate colony or construct <br>
. miniprep candidate colony or construct <br>
-
. Restrict check pSB1C3-TAs/CRs constructs <br>
+
. restriction check pSB3K3-TAs/CRs constructs <br>
-
. Re-Re-ligation trasnformation <br>
+
. Re-Re-ligation transformation <br>
-
. Re-Re-ligation trasnformation V.X <br>
+
. Re-Re-ligation transformation V.X <br>
. Gibson assembly check <br>
. Gibson assembly check <br>
Line 1,516: Line 1,516:
   <div>
   <div>
-
       <a href="#" class="head">Week 4</a>
+
       <a href="#" class="head">Week 5</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>
Wet lab<br>
Wet lab<br>
-
. oligo phosphorilation <br>
+
. oligo phosphorylation <br>
. oligo annealing <br>
. oligo annealing <br>
. ligation of annealed oligos O/N <br>
. ligation of annealed oligos O/N <br>
. gel for C. PCR <br>
. gel for C. PCR <br>
. receptor backbone for annealed oligos <br>
. receptor backbone for annealed oligos <br>
-
. innoculate last ligation <br>
+
. inoculate last ligation <br>
. Check past locks/keys status <br>
. Check past locks/keys status <br>
. P<sub>bad</sub> 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 1,534: Line 1,534:
<br> <b> July 29 </b> <br>
<br> <b> July 29 </b> <br>
Wet lab<br>
Wet lab<br>
-
. Miniprep and restrict check innoculations <br>
+
. Miniprep and restriction check inoculations <br>
-
. Restrict check pSB1C3-TAs/CRs constructs <br>
+
. restriction check pSB3K3-TAs/CRs constructs <br>
. Inoculation of CR and TA from distribution kit <br>
. Inoculation of CR and TA from distribution kit <br>
-
. prepare I20260 in PSB1C3 <br>
+
. prepare BBa_I20260 in pSB3K3 <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>
. Check digested GFP <br>
. Check digested GFP <br>
-
. Check digested B0015 <br>
+
. Check digested BBa_B0015 <br>
. ligate oligos into GFP <br>
. ligate oligos into GFP <br>
-
. ligate oligos itno B0015 <br>
+
. ligate oligos into BBa_B0015 <br>
. prepare M9 for characterization <br>
. prepare M9 for characterization <br>
. 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 pSB1C3 ( with a control) <br>
+
. transformation of Clon001-010 in pSB3K3 ( 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 pSB1C3 (B0015, I13401) <br>
+
. transformation of CLON001-010 in pSB3K3 (BBa_B0015, BBa_I13401) <br>
-
. C. PCR for yesterday transfomations (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 I20260 in PSB1C3 <br>
+
. prepare BBa_I20260 in pSB3K3 <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 1,564:
. 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>
-
. Sequencing preparation for posible construct <br>
+
. Sequencing preparation for possible construct <br>
. colony PCR for 5th <br>
. colony PCR for 5th <br>
. inoculate candidates <br>
. inoculate candidates <br>
-
. prepare i20260in psb1c3 <br>
+
. prepare BBa_I20260 in pSB3K3 <br>
Line 1,604: Line 1,604:
. miniprep CLON005 <br>
. miniprep CLON005 <br>
. Digest 005 Spe1 and Pst1 <br>
. Digest 005 Spe1 and Pst1 <br>
-
. ligate CLON 005 with B0015 <br>
+
. ligate CLON 005 with BBa_B0015 <br>
-
. prepare backbone : B0015 E, X /// I13401 E,X /// J23102 X, P <br>
+
. prepare backbone : BBa_B0015 E, X /// BBa_I13401 E,X /// BBa_J23102 X, P <br>
. Ligate P<sub>bad</sub> with Key1 dTT <br>
. Ligate P<sub>bad</sub> with Key1 dTT <br>
-
. digest ptet lock 1 with S and P <br>
+
. digest P<sub>tet</sub> lock 1 with S and P <br>
<br><b>Aug 2</b> <br>
<br><b>Aug 2</b> <br>
Line 1,626: Line 1,626:
. Restrict P<sub>bad</sub> 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 BBa_B0015 <br>
. Ligate P<sub>bad</sub>.TA <br>
. Ligate P<sub>bad</sub>.TA <br>
-
. liagte CR.GFP.dTT <br>
+
. ligate CR.GFP.dTT <br>
. Ligate P<sub>bad</sub>.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>
. Sequence 001-010 <br>
. Sequence 001-010 <br>
-
. inoculate B0015 <br>
+
. inoculate BBa_B0015 <br>
. inoculate constitutive promoter <br>
. inoculate constitutive promoter <br>
-
. Innoculate P<sub>bad</sub> with key 1 dTT <br>
+
. inoculate 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 P<sub>bad</sub> promoter <br>
. Prepare M9 minimal Medium <br>
. Prepare M9 minimal Medium <br>
. make a second list of oligos to be sent out <br>
. make a second list of oligos to be sent out <br>
. Test out GA mastermix efficiency <br>
. Test out GA mastermix efficiency <br>
-
. Ligate TAs oligo with B0015 <br>
+
. Ligate TAs oligo with BBa_B0015 <br>
. inoue Buffer <br>
. inoue Buffer <br>
. Sequence 001-010 <br>
. Sequence 001-010 <br>
Line 1,649: Line 1,649:
. Colony PCR for TA.dTTs <br>
. Colony PCR for TA.dTTs <br>
. inoculate TA.dTT <br>
. inoculate TA.dTT <br>
-
. ligation of pBAD and key3 <br>
+
. ligation of P<sub>bad</sub> and key3 <br>
. 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>
-
. Ligate Pbad.TA <br>
+
. Ligate P<sub>bad</sub>.TA <br>
-
. liagte CR.GFP.dTT <br>
+
. ligate CR.GFP.dTT <br>
-
. Ligate Pbad.dTT <br>
+
. Ligate P<sub>bad</sub>.dTT <br>
. Transform TAs, CRs <br>
. Transform TAs, CRs <br>
. Prepare M9 medium <br>
. Prepare M9 medium <br>
-
. Prepare Innoculations for Pbad characteriation <br>
+
. Prepare inoculations for P<sub>bad</sub> characterization <br>
-
. Sequecing preapraion <br>
+
. Sequecing preparation <br>
-
. Digest Ptet lock 1 with S and P <br>
+
. Digest P<sub>tet</sub> lock 1 with S and P <br>
. Glycerol stock for 001-010 <br>
. Glycerol stock for 001-010 <br>
. Inoue competent cells <br>
. Inoue competent cells <br>
-
. "Mini Prep pSB1C3-PCRPbad<br>
+
. "Mini Prep pSB3K3-PCRP<sub>bad</sub><br>
<br><b>Aug 6</b> <br>
<br><b>Aug 6</b> <br>
Line 1,673: Line 1,673:
. Restrict TAs <br>
. Restrict TAs <br>
. Restrict CRs <br>
. Restrict CRs <br>
-
. Restrict B0015 for TAs <br>
+
. Restrict BBa_B0015 for TAs <br>
. Colony PCR for TA.dTTs <br>
. Colony PCR for TA.dTTs <br>
. Oligo annealing for TA.dTT <br>
. Oligo annealing for TA.dTT <br>
. Inoue competent cells <br>
. Inoue competent cells <br>
. Sequencing results <br>
. Sequencing results <br>
-
. Ligate PBad with Key1 dTT <br>
+
. Ligate P<sub>bad</sub> with Key1 dTT <br>
-
. Ligate Ptet Lock 1 (GFP dTT) <br>
+
. Ligate P<sub>tet</sub> Lock 1 (GFP dTT) <br>
-
. Send PCR Pbad for sequencing <br>
+
. Send PCR P<sub>bad</sub> for sequencing <br>
-
. Assay Pbad promoter <br>
+
. Assay P<sub>bad</sub> promoter <br>
<br><b>Aug 7</b> <br>
<br><b>Aug 7</b> <br>
Wet lab<br>
Wet lab<br>
. Gel check for C. PCR <br>
. Gel check for C. PCR <br>
-
. inculate possible TA.dTT <br>
+
. inoculate possible TA.dTT <br>
. Ligation of TA and dTT (4TAs) <br>
. Ligation of TA and dTT (4TAs) <br>
-
. Ligation of pBAD and key dTT <br>
+
. Ligation of P<sub>bad</sub> and key dTT <br>
. Gel check for TAs CRs <br>
. Gel check for TAs CRs <br>
. competent cells <br>
. competent cells <br>
-
. miniprep pBAD-dTT <br>
+
. miniprep P<sub>bad</sub>-dTT <br>
-
. miniprep pBAD-key3 <br>
+
. miniprep P<sub>bad</sub>-key3 <br>
-
. Colony PCR for pBAD-dTT and pBAD-key3 <br>
+
. Colony PCR for P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3 <br>
-
. Restriction of pBAD-dTT and pBAD-key3 <br>
+
. Restriction of P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3 <br>
-
. prepare backbone and or insert if any // and restrict check <br>
+
. prepare backbone and or insert if any // and restriction check <br>
-
. Preparation for Pbad promoter characterization <br>
+
. Preparation for P<sub>bad</sub> promoter characterization <br>
-
. Colony PCR for Pbad with Key 1 dTT <br>
+
. Colony PCR for P<sub>bad</sub> with Key 1 dTT <br>
-
. Colony PCR for Ptet Lock 1 GFP dTT <br>
+
. Colony PCR for P<sub>tet</sub> Lock 1 GFP dTT <br>
-
. Innoculation of ligated DNA <br>
+
. inoculation of ligated DNA <br>
<br><b>Aug 8</b> <br>
<br><b>Aug 8</b> <br>
Wet lab<br>
Wet lab<br>
-
. digestion of pBAD-key3 <br>
+
. digestion of P<sub>bad</sub>-key3 <br>
-
. Restriction check for pBAD-dTT and pBAD-key3-dtt <br>
+
. Restriction check for P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3-DTT <br>
-
. ligation of pBAD-key3-dTT <br>
+
. ligation of P<sub>bad</sub>-key3-dTT <br>
. Miniprep for CLON007(J), CLON 010(I) and CLON (G) <br>
. Miniprep for CLON007(J), CLON 010(I) and CLON (G) <br>
-
. Miniprep Pbad Key 1 dTT <br>
+
. Miniprep P<sub>bad</sub> Key 1 dTT <br>
-
. Miniprep Ptet Lock 1 GFP dTT <br>
+
. Miniprep P<sub>tet</sub> Lock 1 GFP dTT <br>
. Restriction Check for the key 1 and lock 1 mentioned above <br>
. Restriction Check for the key 1 and lock 1 mentioned above <br>
. Prepare digestion of key 1 and lock 1 for ligation <br>
. Prepare digestion of key 1 and lock 1 for ligation <br>
Line 1,714: Line 1,714:
. Restriction check CLON007(J), CLON 010(I) and CLON (G) <br>
. Restriction check CLON007(J), CLON 010(I) and CLON (G) <br>
. digestion of CLON007(J), CLON 010(I) and CLON (G) <br>
. digestion of CLON007(J), CLON 010(I) and CLON (G) <br>
-
. ligation of CLON007(J), CLON 010(I) and CLON (G) with pBAD <br>
+
. ligation of CLON007(J), CLON 010(I) and CLON (G) with P<sub>bad</sub> <br>
. C. PCR for CLON 022-025 TAs.dTT <br>
. C. PCR for CLON 022-025 TAs.dTT <br>
-
. ligation of pBAD and TAs <br>
+
. ligation of P<sub>bad</sub> and TAs <br>
. ligation of GFP and CRs <br>
. ligation of GFP and CRs <br>
. Inoue competent cells <br>
. Inoue competent cells <br>
-
. Pbad promoter characterization <br>
+
. P<sub>bad</sub> promoter characterization <br>
<br><b>Aug 9</b> <br>
<br><b>Aug 9</b> <br>
Wet lab<br>
Wet lab<br>
-
. transformation , all in chl, overnight ligation 16C <br>
+
. transformation , all in CHL, overnight ligation 16C <br>
. Colony PCR gel check <br>
. Colony PCR gel check <br>
. Check competent cells <br>
. Check competent cells <br>
Line 1,744: Line 1,744:
. Gel check for C. PCR TAs.dTT <br>
. Gel check for C. PCR TAs.dTT <br>
. Colony PCR for CR.GFP.dTTs <br>
. Colony PCR for CR.GFP.dTTs <br>
-
. Colony PCR for Pbad.TAs <br>
+
. Colony PCR for P<sub>bad</sub>.TAs <br>
. Inoculate CR.GFP.dTTs <br>
. Inoculate CR.GFP.dTTs <br>
-
. Inoculate Pbad.TAs <br>
+
. Inoculate P<sub>bad</sub>.TAs <br>
. Restrict TA.dTTs <br>
. Restrict TA.dTTs <br>
-
. Ligate Pbad.TA.dTTs <br>
+
. Ligate P<sub>bad</sub>.TA.dTTs <br>
-
. Transform Pbad.TA.dTTs <br>
+
. Transform P<sub>bad</sub>.TA.dTTs <br>
-
. Restriction check for Ptet Lock 1 GFP dTT and Pbad Key 1 dTT <br>
+
. Restriction check for P<sub>tet</sub> Lock 1 GFP dTT and P<sub>bad</sub> Key 1 dTT <br>
. Transform 2014 Kit plate 4 well GF <br>
. Transform 2014 Kit plate 4 well GF <br>
. Transformation of restricted checked DNA <br>
. Transformation of restricted checked DNA <br>
-
. Innoculate BBa_J13002 <br>
+
. inoculate BBa_J13002 <br>
-
. Innoculate more Pbad dTTs <br>
+
. inoculate more P<sub>bad</sub> dTTs <br>
. sequencing PCR for MCR, MTA <br>
. sequencing PCR for MCR, MTA <br>
. transformation for backbone with KAN resistant <br>
. transformation for backbone with KAN resistant <br>
. inoculation B0034 from glycerol stock B0034 <br>
. inoculation B0034 from glycerol stock B0034 <br>
. restriction check <br>
. restriction check <br>
-
. Ligation of pBAD-key3 into B0015-pSB1AK3 plasmid <br>
+
. Ligation of P<sub>bad</sub>-key3 into BBa_B0015-pSB1AK3 plasmid <br>
-
. inoculation of pBAD-key3 <br>
+
. inoculation of P<sub>bad</sub>-key3 <br>
<br><b>Aug 12</b> <br>
<br><b>Aug 12</b> <br>
Wet lab<br>
Wet lab<br>
-
. transformation pBAD -TA <br>
+
. transformation P<sub>bad</sub> -TA <br>
. Gel for TA.dTTs <br>
. Gel for TA.dTTs <br>
. GEl for C. PCR for CR.GFP.dTT <br>
. GEl for C. PCR for CR.GFP.dTT <br>
-
. Digestion of PCR pBAD <br>
+
. Digestion of PCR P<sub>bad</sub> <br>
<br><b>Aug 13</b> <br>
<br><b>Aug 13</b> <br>
Wet lab<br>
Wet lab<br>
. miniprep CR.GFP.dTTs <br>
. miniprep CR.GFP.dTTs <br>
-
. miniprep Pbad <br>
+
. miniprep P<sub>bad</sub> <br>
-
. Restrict check CR.GFP.dTTs <br>
+
. restriction check CR.GFP.dTTs <br>
-
. innoculate TA.dTTs <br>
+
. inoculate TA.dTTs <br>
. Gel extraction-purification of constitutive promoter <br>
. Gel extraction-purification of constitutive promoter <br>
-
. Ligate CR.GFP.dTTs into Cosntritutive promoter <br>
+
. Ligate CR.GFP.dTTs into constitutive promoter <br>  
-
. transform, Pbad.Key3 <br>
+
. transform, P<sub>bad</sub>.Key3 <br>
-
. transform Cosntitutive promoter.CR.GFP.dTTs <br>
+
. transform constitutive promoter.CR.GFP.dTTs <br>
. Gibson Assembly mastermix <br>
. Gibson Assembly mastermix <br>
-
. Digest Pbad Key dTT and Ptet Lock 1 GFP dTT for 3A assembly <br>
+
. Digest P<sub>bad</sub> Key dTT and P<sub>tet</sub> Lock 1 GFP dTT for 3A assembly <br>
. Miniprep pSB3K3-J04550 for pSB3K3 Backbone <br>
. Miniprep pSB3K3-J04550 for pSB3K3 Backbone <br>
. 3A assembly <br>
. 3A assembly <br>
-
. Plan for Pbad characterization <br>
+
. Plan for P<sub>bad</sub> characterization <br>
. Prepare stock arabinose solution M9 medium <br>
. Prepare stock arabinose solution M9 medium <br>
Line 1,791: Line 1,791:
. check dNTPs <br>
. check dNTPs <br>
. miniprep TA.dTTs <br>
. miniprep TA.dTTs <br>
-
. Restrict check TA.dTTs <br>
+
. restriction check TA.dTTs <br>
-
. miniprep Lock3.gfp.dTT <br>
+
. miniprep Lock3.GFP.dTT <br>
-
. colony PCR for PBAD-key3-dTT <br>
+
. colony PCR for P<sub>bad</sub>-key3-dTT <br>
. Gel extraction-purification of constitutive promoter <br>
. Gel extraction-purification of constitutive promoter <br>
-
. Ligate CR.GFP.dTTs into Cosntritutive promoter <br>
+
. Ligate CR.GFP.dTTs into constitutive promoter <br>
-
. ligate TA.dTT with Pbad <br>
+
. ligate TA.dTT with P<sub>bad</sub> <br>
-
. transform ligate Pbad.TA.dTTs <br>
+
. transform ligate P<sub>bad</sub>.TA.dTTs <br>
-
. transform Cosntitutive promoter.CR.GFP.dTTs <br>
+
. transform constitutive promoter.CR.GFP.dTTs <br>
<br><b>Aug 15</b> <br>
<br><b>Aug 15</b> <br>
Line 1,804: Line 1,804:
. Gibson Assembly mastermix <br>
. Gibson Assembly mastermix <br>
. purify CR.GFP.dTTs <br>
. purify CR.GFP.dTTs <br>
-
. Ligate CR.GFP.dTTs into Cosntritutive promoter <br>
+
. Ligate CR.GFP.dTTs into constitutive promoter <br>
. Digestion of TA-dTT and PCR clean-up <br>
. Digestion of TA-dTT and PCR clean-up <br>
. Restriction Check for TA-dTT <br>
. Restriction Check for TA-dTT <br>
-
. transform ligate Pbad.TA.dTTs <br>
+
. transform ligate P<sub>bad</sub>.TA.dTTs <br>
-
. transform Cosntitutive promoter.CR.GFP.dTTs <br>
+
. transform constitutive promoter.CR.GFP.dTTs <br>
. make 10x DNA loading Dye <br>
. make 10x DNA loading Dye <br>
. Order new TA and CR <br>
. Order new TA and CR <br>
Line 1,827: Line 1,827:
<br><b>Aug 18</b> <br>
<br><b>Aug 18</b> <br>
Wet lab<br>
Wet lab<br>
-
. Restriction check for Pbad.TA.dTTs <br>
+
. Restriction check for P<sub>bad</sub>.TA.dTTs <br>
. Restriction check for constitutive promoter.CR.GFP.dTT <br>
. Restriction check for constitutive promoter.CR.GFP.dTT <br>
. Restriction check for constitutive promoter.RBS.GFP.dTT <br>
. Restriction check for constitutive promoter.RBS.GFP.dTT <br>
Line 1,834: Line 1,834:
. PCR check for constitutive promoter.CR.GFP.dTT <br>
. PCR check for constitutive promoter.CR.GFP.dTT <br>
. PCR check for constitutive promoter.RBS.GFP.dTT <br>
. PCR check for constitutive promoter.RBS.GFP.dTT <br>
-
. PCR check for Pbad.TA.dTT <br>
+
. PCR check for P<sub>bad</sub>.TA.dTT <br>
-
. Restrict check TA.dTTs <br>
+
. restriction check TA.dTTs <br>
. Prepare functional assay for RBS, CRs <br>
. Prepare functional assay for RBS, CRs <br>
. Inoculate candidates <br>
. Inoculate candidates <br>
. Prepare pSB3K3 <br>
. Prepare pSB3K3 <br>
-
. Prepare pSB1C3 for oligo ligation <br>
+
. Prepare pSB3K3 for oligo ligation <br>
-
. Prepare I13401 for oligo ligation <br>
+
. Prepare BBa_I13401 for oligo ligation <br>
-
. Prepare B0015 for oligo ligation <br>
+
. Prepare BBa_B0015 for oligo ligation <br>
. Inoue competent cells <br>
. Inoue competent cells <br>
-
. Prepare for Pbad characterization <br>
+
. Prepare for P<sub>bad</sub> characterization <br>
-
. Take over the tank B and C arround 1pm-5;30 <br>
+
. Take over the tank B and C around 1pm-5;30 <br>
<br><b>Aug 19</b> <br>
<br><b>Aug 19</b> <br>
Line 1,853: Line 1,853:
. miniprep possible candidates <br>
. miniprep possible candidates <br>
. Restrict TA.dTT <br>
. Restrict TA.dTT <br>
-
. pSB1C3-I13401 for oligo ligation <br>
+
. pSB3K3-BBa_I13401 for oligo ligation <br>
-
. B0015 for oligo ligation <br>
+
. BBa_B0015 for oligo ligation <br>
-
. pSB1C3-J04450 for oligo ligation <br>
+
. pSB3K3-BBa_J04450 for oligo ligation <br>
. Inoue competent cells <br>
. Inoue competent cells <br>
-
. pBAD-key3-dTT restriction check <br>
+
. P<sub>bad</sub>-key3-dTT restriction check <br>
<br><b>Aug 20</b> <br>
<br><b>Aug 20</b> <br>
Wet lab<br>
Wet lab<br>
-
. Gel check for Pbad.TA.dTTs <br>
+
. Gel check for P<sub>bad</sub>.TA.dTTs <br>
-
. purification for B0015, I13401 <br>
+
. purification for BBa_B0015, BBa_I13401 <br>
-
. pSB1C3-J04450 for oligo ligation <br>
+
. pSB3K3-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 J23102, LOCK 3 GFP dtt <br>
+
. PCR check for BBa_J23102, LOCK 3 GFP DTT <br>
-
. restrcition check for key1 dtt <br>
+
. Restriction check for key1 DTT <br>
<br><b>Aug 21</b> <br>
<br><b>Aug 21</b> <br>
Line 1,873: Line 1,873:
. Ligate UST lock <br>
. Ligate UST lock <br>
. restriction check for key1.dTT <br>
. restriction check for key1.dTT <br>
-
. ligate pbad.key1.dTT <br>
+
. ligate P<sub>bad</sub>.key1.dTT <br>
. ligate lock 3.GFP.dTT with C. promoter <br>
. ligate lock 3.GFP.dTT with C. promoter <br>
. Colony PCR for c. promoter.MCR.GFP.dTT <br>
. Colony PCR for c. promoter.MCR.GFP.dTT <br>
Line 1,879: Line 1,879:
. Glycerol stocks <br>
. Glycerol stocks <br>
. transform HKUSTlock.GFP.dTT <br>
. transform HKUSTlock.GFP.dTT <br>
-
. Transform pSB1C3-HKUSTlock <br>
+
. Transform pSB3K3-HKUSTlock <br>
Line 1,885: Line 1,885:
Wet lab<br>
Wet lab<br>
. competent cells <br>
. competent cells <br>
-
. Colony PCR for pSB1C3-HKUSTlock1 <br>
+
. Colony PCR for pSB3K3-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>
. restrict pSB3K3 for 3 pieces assemblage <br>
. restrict pSB3K3 for 3 pieces assemblage <br>
. ligate TA-CR into pSB3K3 <br>
. ligate TA-CR into pSB3K3 <br>
. Transform TA-CR <br>
. Transform TA-CR <br>
-
. colony PCR for PBAD-key3-dTT <br>
+
. colony PCR for P<sub>bad</sub>-key3-dTT <br>
-
. inoculate Pbad.key1.dTT <br>
+
. inoculate P<sub>bad</sub>.key1.dTT <br>
. gel check for C. PCR on 21/08 <br>
. gel check for C. PCR on 21/08 <br>
. extraction of pSB3K3 <br>
. extraction of pSB3K3 <br>
. inoculate HKUSTlock1.GFP.dTT <br>
. inoculate HKUSTlock1.GFP.dTT <br>
-
. inoculate pSB1C3-HKUSTlock1 <br>
+
. inoculate pSB3K3-HKUSTlock1 <br>
Line 1,902: Line 1,902:
Wet lab<br>
Wet lab<br>
. Gel check for TAs <br>
. Gel check for TAs <br>
-
. colony PCR for UST lock in I13401 and pSB1C3 <br>
+
. colony PCR for UST lock in BBa_I13401 and pSB3K3 <br>
. transform ust key <br>
. transform ust key <br>
. Check competent cells <br>
. Check competent cells <br>
Line 1,910: Line 1,910:
<br><b>Aug 24</b> <br>
<br><b>Aug 24</b> <br>
Wet lab<br>
Wet lab<br>
-
. Colony PCR for Pbad.Key1.dTT <br>
+
. Colony PCR for P<sub>bad</sub>.Key1.dTT <br>
-
. Gel check for Pbad.Key1.dTT <br>
+
. Gel check for P<sub>bad</sub>.Key1.dTT <br>
Line 1,922: Line 1,922:
<br><b>Aug 25</b> <br>
<br><b>Aug 25</b> <br>
Wet lab<br>
Wet lab<br>
-
. Colony PCR for Pbad.Key1.dTT <br>
+
. Colony PCR for P<sub>bad</sub>.Key1.dTT <br>
. PCR check for CR.GFP.dTT <br>
. PCR check for CR.GFP.dTT <br>
. PCR check for Cons. prom.CR.GFP.dTT <br>
. PCR check for Cons. prom.CR.GFP.dTT <br>
Line 1,929: Line 1,929:
. transform ust lock1 <br>
. transform ust lock1 <br>
. transform ust key <br>
. transform ust key <br>
-
. ligation again key 1 into B0015 <br>
+
. ligation again key 1 into BBa_B0015 <br>
-
. Double primer PCR for comD fragments (mutagenesis) for Gibson Assembly<br>
+
Line 1,939: Line 1,938:
. Colony PCR for "un-named key" <br>
. Colony PCR for "un-named key" <br>
. inoculate ones which SEEMS like it contains key <br>
. inoculate ones which SEEMS like it contains key <br>
-
. colony pcr for Key1.dtt <br>
+
. colony PCR for Key1.DTT <br>
. Gel check for O/NPCR products <br>
. Gel check for O/NPCR products <br>
-
. 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 I13401 for CRs <br>
+
. Feeling paranoic? Prepare BBa_I13401 for CRs <br>
-
. Streak posible 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 5 alpha cell <br>
+
. make DH5alpha cell <br>
Line 1,954: Line 1,953:
Wet lab<br>
Wet lab<br>
. miniprerp candidates <br>
. miniprerp candidates <br>
-
. pcr check for candidates <br>
+
. PCR check for candidates <br>
-
. gel check for o/n pcr <br>
+
. gel check for o/n PCR <br>
-
. restrict check for ust key 1 <br>
+
. restriction check for ust key 1 <br>
. restrick check for key 1 <br>
. restrick check for key 1 <br>
-
. Pbad for ust key 1 <br>
+
. P<sub>bad</sub> for ust key 1 <br>
-
. extract ust lock 1 for j23102 <br>
+
. extract ust lock 1 for BBa_J23102 <br>
. ligate ust lock and key <br>
. ligate ust lock and key <br>
. 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 1,969: Line 1,968:
. plasmid PCR for HKUSt lock and key <br>
. plasmid PCR for HKUSt lock and key <br>
. 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-BBa_B0015 <br>
Line 1,977: Line 1,976:
. miniprep locks <br>
. miniprep locks <br>
. glycerol stocks for locks <br>
. glycerol stocks for locks <br>
-
. minipreps for B0015 and B0034 <br>
+
. minipreps for BBa_B0015 and B0034 <br>
-
. ligation of pBAD-Hkust key-B0015 <br>
+
. ligation of P<sub>bad</sub>-Hkust key-BBa_B0015 <br>
. digestion of HKUST lock <br>
. digestion of HKUST lock <br>
. ligation of HKUST lock with GFP <br>
. ligation of HKUST lock with GFP <br>
Line 1,986: Line 1,985:
. inoculation of key and lock <br>
. inoculation of key and lock <br>
. miniprep <br>
. miniprep <br>
-
. Key one restrict check <br>
+
. Key one restriction check <br>
-
. key one restriction for inserting pBAD <br>
+
. key one restriction for inserting P<sub>bad</sub> <br>
. Restrict and exytact lock3c rbs and ligate into c.promoter <br>
. Restrict and exytact 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. promoterlock 3c rbs.GFP.DTT <br>
. Extract mcr/mta <br>
. Extract mcr/mta <br>
-
. ligate MCR and MTA into 3K3 <br>
+
. ligate MCR and MTA into pSB3K3 <br>
. transform MCR/MTA <br>
. transform MCR/MTA <br>
. Glycerol stock for locks <br>
. Glycerol stock for locks <br>
-
. 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 aneling for key 1 <br>
Line 2,008: Line 2,007:
. competent cells <br>
. competent cells <br>
. Extract mcr/mta <br>
. Extract mcr/mta <br>
-
. ligate MCR and MTA into 3K3 <br>
+
. ligate MCR and MTA into pSB3K3 <br>
. 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>
-
. Restriction check for pSB1C3-comE and pSB1C3-comED58E<br>
 
Line 2,050: Line 2,048:
<br><b>Sept 1</b> <br>
<br><b>Sept 1</b> <br>
Wet lab<br>
Wet lab<br>
-
. restrict check pBAD- Lost your key-dtt <br>
+
. restriction check P<sub>bad</sub>- Lost your key-DTT <br>
-
. restrict check pBAD-Lost your key-dtt <br>
+
. restriction check P<sub>bad</sub>-Lost your key-DTT <br>
. colony PCR for CR.TAs (orthogonality) <br>
. colony PCR for CR.TAs (orthogonality) <br>
-
. colony PCR for Key1,dtt <br>
+
. colony PCR for Key1,DTT <br>
. ligate lock3c.GFP.dTT into c.p plasmid <br>
. ligate lock3c.GFP.dTT into c.p plasmid <br>
-
. transform lock3.gfp.dtt <br>
+
. transform lock3.GFP.DTT <br>
. 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>
-
. restriciton check for Lost Your Key in PSB1C3 <br>
+
. restriction check for Lost Your Key in pSB3K3 <br>
-
. transform pbad.lost your key.dtt <br>
+
. transform P<sub>bad</sub>.lost your key.DTT <br>
. inoculate orthogonality candidates <br>
. inoculate orthogonality candidates <br>
-
. Prepara midi-prep for 3k3 <br>
+
. Prepare midi-prep for pSB3K3 <br>
Line 2,068: Line 2,066:
<br><b>Sept 2</b> <br>
<br><b>Sept 2</b> <br>
Wet lab<br>
Wet lab<br>
-
. Gel check for O/N colony pcr for key1 and 3k3 <br>
+
. Gel check for O/N colony PCR for key1 and pSB3K3 <br>
. massive prep <br>
. massive prep <br>
. PCR check sense reverse <br>
. PCR check sense reverse <br>
-
. colony pcr for orthogonality <br>
+
. colony PCR for orthogonality <br>
-
. colony pcr for cons. pro. lock3crbs.gfp.dtt <br>
+
. colony PCR for cons. pro. lock3crbs.GFP.DTT <br>
-
. migrate cpromoter E0240 to psb3k3 with mta key <br>
+
. migrate cpromoter BBa_E0240 to pSB3K3 with mta key <br>
-
. 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 pSB1C3 <br>
+
. sequencing for sherlock in pSB3K3 <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 2,085: Line 2,083:
<br><b>Sept 3</b> <br>
<br><b>Sept 3</b> <br>
Wet lab<br>
Wet lab<br>
-
. digestion of Pbad MTA dTT <br>
+
. digestion of P<sub>bad</sub> MTA dTT <br>
-
. digestion of Pbad dTT <br>
+
. digestion of P<sub>bad</sub> dTT <br>
-
. digestion of J23102 MCR GFP dTT <br>
+
. digestion of BBa_J23102 MCR GFP dTT <br>
-
. digestion of J23102 E0240(B0032 GFP dTT) <br>
+
. digestion of BBa_J23102 BBa_E0240(B0032 GFP dTT) <br>
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450) <br>
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450) <br>
-
. Ligation of J23102 MCR GFP dTT with Pbad dTT <br>
+
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
-
. Ligation of J23102 E0240 with Pbad MTA dTT <br>
+
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br>
-
. Ligation of J23102 E0240 with Pbad dTT <br>
+
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> dTT <br>
-
. Plasmid PCR for SherLock-GFP-dtt <br>
+
. Plasmid PCR for SherLock-GFP-DTT <br>
-
. ligation of B0034-GFP-dtt <br>
+
. ligation of B0034-GFP-DTT <br>
-
. ligation of SherLock-GFP-dtt <br>
+
. ligation of SherLock-GFP-DTT <br>
. sequencing <br>
. sequencing <br>
. check plate for arabinose induced mcr mta while singing <br>
. check plate for arabinose induced mcr mta while singing <br>
. extraction of key and lock <br>
. extraction of key and lock <br>
. restriction check for key with lock <br>
. restriction check for key with lock <br>
-
. extration of key1 dtt <br>
+
. extraction of key1 DTT <br>
-
. ligation of key1 dtt with pBAD <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>
+
. CRAZY 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>
. transform lock3c RBS.GFP.dTT <br>
. transform lock3c RBS.GFP.dTT <br>
-
. Midi prep preparations <br>
+
. Midi-prep preparations <br>
-
. Gibson Assembly for comD fragments<br>
+
Line 2,116: Line 2,113:
Wet lab<br>
Wet lab<br>
. Gel purification of digested insert and backbone <br>
. Gel purification of digested insert and backbone <br>
-
. Ligation of J23102 MCR GFP dTT with Pbad dTT <br>
+
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
-
. Ligation of J23102 E0240 with Pbad MTA dTT <br>
+
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br>
-
. digestion of J23102 E0240(B0032 GFP dTT) <br>
+
. digestion of BBa_J23102 BBa_E0240(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>
-
. Colony PCR for B0034-GFP-dtt <br>
+
. Colony PCR for B0034-GFP-DTT <br>
. 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>
Line 2,134: Line 2,131:
<br><b>Sept 5</b> <br>
<br><b>Sept 5</b> <br>
Wet lab<br>
Wet lab<br>
-
. miniprep/glycerol stocck <br>
+
. miniprep/glycerol stock <br>
-
. Transform Pbad dTT <br>
+
. Transform P<sub>bad</sub> dTT <br>
-
. Transform ligated produce of J23102 MCR GFP dTT with Pbad dTT <br>
+
. Transform ligated produce of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
-
. Restriction check for pSB1C3-comD<br>
+
Line 2,150: Line 2,146:
<br><b>Sept 8</b> <br>
<br><b>Sept 8</b> <br>
Wet lab<br>
Wet lab<br>
-
. Repeat mutagenesis for comD second site<br>
+
·      <br>
 +
·      <br>
 +
·      <br>
 +
·   <br>
 +
·      <br>
 +
·      <br>
 +
·      <br>
Line 2,165: Line 2,167:
<br><b>Sept 10</b> <br>
<br><b>Sept 10</b> <br>
Wet lab<br>
Wet lab<br>
-
. colony pcr <br>
+
. colony PCR <br>
. ligations for final constructs <br>
. ligations for final constructs <br>
. inoculations <br>
. inoculations <br>
-
. Pbad.lost your key.dtt <br>
+
. P<sub>bad</sub>.lost your key.DTT <br>
. sherlock <br>
. sherlock <br>
-
. gel check for colony pcr <br>
+
. gel check for colony PCR <br>
-
. colony pcr <br>
+
. colony PCR <br>
Line 2,185: Line 2,187:
<br><b>Sept 12</b> <br>
<br><b>Sept 12</b> <br>
Wet lab<br>
Wet lab<br>
-
. colony pcr <br>
+
. colony PCR <br>
-
. restrict key1.dtt for Pbad insertion <br>
+
. restrict key1.DTT for P<sub>bad</sub> insertion <br>
-
. ligate Pbad with key1.dtt <br>
+
. ligate P<sub>bad</sub> with key1.DTT <br>
. transform key1.dTT <br>
. transform key1.dTT <br>
. extract pSB3K3 <br>
. extract pSB3K3 <br>
Line 2,197: Line 2,199:
. inoculation <br>
. inoculation <br>
. Digestion of CLON 036 <br>
. Digestion of CLON 036 <br>
-
. colony pcr mcr mta <br>
+
. colony PCR mcr mta <br>
Line 2,216: Line 2,218:
<br><b>Sept 16</b> <br>
<br><b>Sept 16</b> <br>
Wet lab<br>
Wet lab<br>
-
. mini prep Pbad.key1.dtt <br>
+
. mini prep P<sub>bad</sub>.key1.DTT <br>
-
. restrict check P<sub>bad</sub>.key1.dtt <br>
+
. restriction 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 P<sub>bad</sub>.key1 <br>
. extract P<sub>bad</sub>.key1 <br>
. inoculate every CR.TA construct <br>
. inoculate every CR.TA construct <br>
Line 2,232: Line 2,234:
Wet lab<br>
Wet lab<br>
. purify P<sub>bad</sub>.key1 <br>
. purify P<sub>bad</sub>.key1 <br>
-
. ligate P<sub>bad</sub>key1 dtt with locks <br>
+
. ligate P<sub>bad</sub>key1 DTT with locks <br>
-
. transform locks.P<sub>bad</sub>.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>
Line 2,244: Line 2,246:
. Spread a plate <br>
. Spread a plate <br>
. miniprep for 3 inoculations <br>
. miniprep for 3 inoculations <br>
-
. Sense oligo and VR PCR for HKUST Lock-dtt construct <br>
+
. Sense oligo and VR PCR for HKUST Lock-DTT construct <br>
. Overnight digestion of plasmid which gives a band around 1300bp <br>
. Overnight digestion of plasmid which gives a band around 1300bp <br>
. EtoHpp for sequencing <br>
. EtoHpp for sequencing <br>
-
. Miniprep construtcs for submission <br>
+
. Miniprep constructs for submission <br>
-
. innoculation <br>
+
. inoculation <br>
. transform 031, 034 <br>
. transform 031, 034 <br>
. Check "Diagonal constructs" and controls <br>
. Check "Diagonal constructs" and controls <br>
-
. migrate CR crontruts into 3K3 <br>
+
. migrate CR constructs into pSB3K3 <br>
Line 2,260: Line 2,262:
. miniprep lock.key candidate <br>
. miniprep lock.key candidate <br>
. ligate problematic cr.key <br>
. ligate problematic cr.key <br>
-
. restrick check sherlock <br>
+
. restriction check sherlock <br>
-
. extrations.... madness <br>
+
. extractions.... madness <br>
Line 2,275: Line 2,277:
<br><b>Sept 21</b> <br>
<br><b>Sept 21</b> <br>
Wet lab<br>
Wet lab<br>
-
. miniprep martching CR.TA <br>
+
. miniprep matching CR.TA <br>
. Restriction check by EcoR1 and Pst1 <br>
. Restriction check by EcoR1 and Pst1 <br>
Line 2,289: Line 2,291:
Wet lab<br>
Wet lab<br>
. Run Gel check for matching set of CR.TA <br>
. Run Gel check for matching set of CR.TA <br>
-
. ligate problrmatic sets <br>
+
. ligate problematic sets <br>
-
. transform rpoblematic sets <br>
+
. transform problematic sets <br>
. prepare DNA for submission <br>
. prepare DNA for submission <br>
-
. Gel purfication for Doyle <br>
+
. Gel purification for Doyle <br>
-
. Ligation lock-dtt with cons. promoter <br>
+
. Ligation lock-DTT with constitutive promoter <br>
. transformation <br>
. transformation <br>
-
. pSB3K3- CRs ligation <br>
+
. pSB3K3-CRs ligation <br>
Line 2,302: Line 2,304:
<br><b>Sept 23</b> <br>
<br><b>Sept 23</b> <br>
Wet lab<br>
Wet lab<br>
-
. pSB3K3- CRs transformation <br>
+
. pSB3K3-CRs transformation <br>
. Gel extractions <br>
. Gel extractions <br>
. Gel purifications <br>
. Gel purifications <br>
Line 2,323: Line 2,325:
. colony PCR <br>
. colony PCR <br>
. miniprep for biobrick submission <br>
. miniprep for biobrick submission <br>
-
. send plamsid for sequencing <br>
+
. send plasmid for sequencing <br>
Line 2,340: Line 2,342:
. Gel check PvuII digestions <br>
. Gel check PvuII digestions <br>
. Gel check AseI digestions <br>
. Gel check AseI digestions <br>
-
. 3A assembly for non pSB1C3 <br>
+
. 3A assembly for non pSB3K3 <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 2,354: Line 2,356:
. C. PCR for H series... <br>
. C. PCR for H series... <br>
. inoculate possible candidates <br>
. inoculate possible candidates <br>
-
. check self ligated 3K3 <br>
+
. check self ligated pSB3K3 <br>
. Gel check for possible candidates AseI <br>
. Gel check for possible candidates AseI <br>
. Gel check for possible candidates PvuII <br>
. Gel check for possible candidates PvuII <br>
. Ligated H6 H7 <br>
. Ligated H6 H7 <br>
. transformed H6 H7 <br>
. transformed H6 H7 <br>
-
. O/N innoculation of MCR/MTA for characterization <br>
+
. O/N inoculation of MCR/MTA for characterization <br>
Line 2,365: Line 2,367:
<br><b>Sept 28</b> <br>
<br><b>Sept 28</b> <br>
Wet lab<br>
Wet lab<br>
-
. Extract pSB1C3 <br>
+
. Extract pSB3K3 <br>
-
. Etract 036 <br>
+
. Extract 036 <br>
. miniprep "H" series candidates <br>
. miniprep "H" series candidates <br>
. Repeat CHL restrictions <br>
. Repeat CHL restrictions <br>
-
. Repeat CHL ligattions <br>
+
. Repeat CHL ligations <br>
. Repeat CHL transformations <br>
. Repeat CHL transformations <br>
. Gel check for "7" series using NcoI <br>
. Gel check for "7" series using NcoI <br>
-
. Restrict check AseI <br>
+
. restriction check AseI <br>
-
. Restrict check NcoI <br>
+
. restriction check NcoI <br>
. 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>
Line 2,388: Line 2,390:
<br><b>Sept 29</b> <br>
<br><b>Sept 29</b> <br>
Wet lab<br>
Wet lab<br>
-
. Restrict check AseI <br>
+
. restriction check AseI <br>
-
. Restrict 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>
Line 2,396: Line 2,398:
. 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 pSB1C3 bacbone EcoRI and PstI <br>
+
. Get pSB3K3 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>
-
. Innoculate Lock and Keys for crude functional assay. <br>
+
. inoculate Lock and Keys for crude functional assay. <br>
Line 2,406: Line 2,408:
Wet lab<br>
Wet lab<br>
. miniprep 11 tubes in shaker c <br>
. miniprep 11 tubes in shaker c <br>
-
. Restrict check H" series with NcoI <br>
+
. restriction check H" series with NcoI <br>
-
. Restrict inoculated colonies wiht AseI <br>
+
. Restrict inoculated colonies with AseI <br>
-
. Restrict inoculated colonies wiht NcoI <br>
+
. Restrict inoculated colonies with 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>
-
. transformtation of 16 confirmed constructs <br>
+
. transformation of 16 confirmed constructs <br>
Line 2,417: Line 2,419:
Wet lab<br>
Wet lab<br>
. digestion of key3,HKUSTkey? <br>
. digestion of key3,HKUSTkey? <br>
-
. miniprep- final constructs <br>
+
. miniprep final constructs <br>

Revision as of 14:40, 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.






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 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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