Team:Hong Kong HKUST/wetlab/notebook

From 2014.igem.org

(Difference between revisions)
Line 1,849: Line 1,849:
<br><b>Aug 1</b> <br>
<br><b>Aug 1</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. colony PCR for 5th <br>
-
·      <br>
+
. Digest Biobrick TA and CR <br>
-
·      <br>
+
. short ligation for TAs CRs <br>
-
·   <br>
+
. Transform TAs, CRs <br>
 +
. inoue Buffer <br>
 +
. inoculations if possible <br>
 +
. miniprep CLON005 <br>
 +
. Digest 005 Spe1 and Pst1 <br>
 +
. ligate CLON 005 with B0015 <br>
 +
. prepare backbone : B0015 E, X /// I13401 E,X /// J23102 X, P <br>
 +
. Ligate PBad with Key1 dTT <br>
 +
. digest ptet lock 1 with S and P <br>
 +
 
 +
<br><b>Aug 2</b> <br>
 +
Wet lab<br>
 +
. Devise a general blue print for the catalog and feature page <br>
 +
 
       </div>
       </div>
Line 1,861: Line 1,874:
<br><b>Aug 4</b> <br>
<br><b>Aug 4</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Mini prep 001-010 <br>
-
·      <br>
+
. Restrict TAs <br>
-
·      <br>
+
. restrict CRs <br>
-
·   <br>
+
. Restrict pBAD for TAs <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Restrict B0015 <br>
 +
. Ligate Pbad.TA <br>
 +
. liagte CR.GFP.dTT <br>
 +
. Ligate Pbad.dTT <br>
 +
. Transform TAs, CRs <br>
 +
. inoculate 001-010 <br>
 +
. Sequence 001-010 <br>
 +
. inoculate B0015 <br>
 +
. inoculate constitutive promoter <br>
 +
. Innoculate Pbad with key 1 dTT <br>
 +
. Competent cells preparations DH10B <br>
 +
. Prepare for functional assay of Pbad promoter <br>
 +
. Prepare M9 minimal Medium <br>
 +
. make a second list of oligos to be sent out <br>
 +
. Test out GA mastermix efficiency <br>
 +
. Ligate TAs oligo with B0015 <br>
 +
. inoue Buffer <br>
 +
. Sequence 001-010 <br>
 +
 
<br><b>Aug 5</b> <br>
<br><b>Aug 5</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Colony PCR for TA.dTTs <br>
-
·      <br>
+
. inoculate TA.dTT <br>
-
·      <br>
+
. ligation of pBAD and key3 <br>
-
·   <br>
+
. Restrict TAs <br>
 +
. restrict CRs <br>
 +
. Restrict pBAD for TAs <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Ligate Pbad.TA <br>
 +
. liagte CR.GFP.dTT <br>
 +
. Ligate Pbad.dTT <br>
 +
. Transform TAs, CRs <br>
 +
. Prepare M9 medium <br>
 +
. Prepare Innoculations for Pbad characteriation <br>
 +
. Sequecing preapraion <br>
 +
. Digest Ptet lock 1 with S and P <br>
 +
. Glycerol stock for 001-010 <br>
 +
. Inoue competent cells <br>
 +
. "Mini Prep pSB1C3-PCRPbad<br>
 +
 
<br><b>Aug 6</b> <br>
<br><b>Aug 6</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Miniprep TA.dTT <br>
-
·      <br>
+
. Restriction check for TA.dTT <br>
-
·      <br>
+
. Miniprep TAs CRs <br>
-
·   <br>
+
. Restrict TAs <br>
 +
. Restrict CRs <br>
 +
. Restrict B0015 for TAs <br>
 +
. Colony PCR for TA.dTTs <br>
 +
. Oligo annealing for TA.dTT <br>
 +
. Inoue competent cells <br>
 +
. Sequencing results <br>
 +
. Ligate PBad with Key1 dTT <br>
 +
. Ligate Ptet Lock 1 (GFP dTT) <br>
 +
. Send PCR Pbad for sequencing <br>
 +
. Assay Pbad promoter <br>
 +
 
<br><b>Aug 7</b> <br>
<br><b>Aug 7</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Gel check for C. PCR <br>
-
·      <br>
+
. inculate possible TA.dTT <br>
-
·      <br>
+
. Ligation of TA and dTT (4TAs) <br>
-
·   <br>
+
. Ligation of pBAD and key dTT <br>
 +
. Gel check for TAs CRs <br>
 +
. competent cells <br>
 +
. miniprep pBAD-dTT <br>
 +
. miniprep pBAD-key3 <br>
 +
. Colony PCR for pBAD-dTT and pBAD-key3 <br>
 +
. Restriction of pBAD-dTT and pBAD-key3 <br>
 +
. prepare backbone and or insert if any // and restrict check <br>
 +
. Preparation for Pbad promoter characterization <br>
 +
. Colony PCR for Pbad with Key 1 dTT <br>
 +
. Colony PCR for Ptet Lock 1 GFP dTT <br>
 +
. Innoculation of ligated DNA <br>
 +
 
<br><b>Aug 8</b> <br>
<br><b>Aug 8</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. digestion of pBAD-key3 <br>
-
·      <br>
+
. Restriction check for pBAD-dTT and pBAD-key3-dtt <br>
-
·      <br>
+
. ligation of pBAD-key3-dTT <br>
-
·   <br>
+
. Miniprep for CLON007(J), CLON 010(I) and CLON (G) <br>
 +
. Miniprep Pbad Key 1 dTT <br>
 +
. Miniprep Ptet Lock 1 GFP dTT <br>
 +
. Restriction Check for the key 1 and lock 1 mentioned above <br>
 +
. Prepare digestion of key 1 and lock 1 for ligation <br>
 +
. Transform BBa_J13002 <br>
 +
. Restriction check 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>
 +
. C. PCR for CLON 022-025 TAs.dTT <br>
 +
. ligation of pBAD and TAs <br>
 +
. ligation of GFP and CRs <br>
 +
. Inoue competent cells <br>
 +
. Pbad promoter characterization <br>
 +
 
 +
<br><b>Aug 9</b> <br>
 +
Wet lab<br>
 +
. transformation , all in chl, overnight ligation 16C <br>
 +
. Colony PCR gel check <br>
 +
. Check competent cells <br>
 +
 
 +
<br><b>Aug 10</b> <br>
 +
Wet lab<br>
 +
. Restriction check for TAs.dTT <br>
 +
. Gel check for C. PCR TAs.dTT <br>
 +
 
 +
 
       </div>
       </div>
Line 1,897: Line 1,994:
<br><b>Aug 11</b> <br>
<br><b>Aug 11</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Restriction check for TAs.dTT <br>
-
·      <br>
+
. Gel check for C. PCR TAs.dTT <br>
-
·      <br>
+
. Colony PCR for CR.GFP.dTTs <br>
-
·   <br>
+
. Colony PCR for Pbad.TAs <br>
 +
. Inoculate CR.GFP.dTTs <br>
 +
. Inoculate Pbad.TAs <br>
 +
. Restrict TA.dTTs <br>
 +
. Ligate Pbad.TA.dTTs <br>
 +
. Transform Pbad.TA.dTTs <br>
 +
. Restriction check for Ptet Lock 1 GFP dTT and Pbad Key 1 dTT <br>
 +
. Transform 2014 Kit plate 4 well GF <br>
 +
. Transformation of restricted checked DNA <br>
 +
. Innoculate BBa_J13002 <br>
 +
. Innoculate more Pbad dTTs <br>
 +
. sequencing PCR for MCR, MTA <br>
 +
. transformation for backbone with KAN resistant <br>
 +
. inoculation B0034 from glycerol stock B0034 <br>
 +
. restriction check <br>
 +
. Ligation of pBAD-key3 into B0015-pSB1AK3 plasmid <br>
 +
. inoculation of pBAD-key3 <br>
 +
 
<br><b>Aug 12</b> <br>
<br><b>Aug 12</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. transformation pBAD -TA <br>
-
·      <br>
+
. Gel for TA.dTTs <br>
-
·      <br>
+
. GEl for C. PCR for CR.GFP.dTT <br>
-
·   <br>
+
. Digestion of PCR pBAD <br>
 +
 
<br><b>Aug 13</b> <br>
<br><b>Aug 13</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. miniprep CR.GFP.dTTs <br>
-
·      <br>
+
. miniprep Pbad <br>
-
·      <br>
+
. Restrict check CR.GFP.dTTs <br>
-
·   <br>
+
. innoculate TA.dTTs <br>
 +
. Gel extraction-purification of constitutive promoter <br>
 +
. Ligate CR.GFP.dTTs into Cosntritutive promoter <br>
 +
. transform, Pbad.Key3 <br>
 +
. transform Cosntitutive promoter.CR.GFP.dTTs <br>
 +
. Gibson Assembly mastermix <br>
 +
. Digest Pbad Key dTT and Ptet Lock 1 GFP dTT for 3A assembly <br>
 +
. Miniprep pSB3K3-J04550 for pSB3K3 Backbone <br>
 +
. 3A assembly <br>
 +
. Plan for Pbad characterization <br>
 +
. Prepare stock arabinose solution M9 medium <br>
 +
 
<br><b>Aug 14</b> <br>
<br><b>Aug 14</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Gel check for lock3.GFP.dTT colony PCR I-VI <br>
-
·      <br>
+
. check dNTPs <br>
-
·      <br>
+
. miniprep TA.dTTs <br>
-
·   <br>
+
. Restrict check TA.dTTs <br>
 +
. miniprep Lock3.gfp.dTT <br>
 +
. colony PCR for PBAD-key3-dTT <br>
 +
. Gel extraction-purification of constitutive promoter <br>
 +
. Ligate CR.GFP.dTTs into Cosntritutive promoter <br>
 +
. ligate TA.dTT with Pbad <br>
 +
. transform ligate Pbad.TA.dTTs <br>
 +
. transform Cosntitutive promoter.CR.GFP.dTTs <br>
 +
 
<br><b>Aug 15</b> <br>
<br><b>Aug 15</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Gibson Assembly mastermix <br>
-
·      <br>
+
. purify CR.GFP.dTTs <br>
-
·      <br>
+
. Ligate CR.GFP.dTTs into Cosntritutive promoter <br>
-
·   <br>
+
. Digestion of TA-dTT and PCR clean-up <br>
 +
. Restriction Check for TA-dTT <br>
 +
. transform ligate Pbad.TA.dTTs <br>
 +
. transform Cosntitutive promoter.CR.GFP.dTTs <br>
 +
. make 10x DNA loading Dye <br>
 +
. Order new TA and CR <br>
 +
. Gibson Assembly mastermix <br>
 +
 
 +
<br><b>Aug 16</b> <br>
 +
Wet lab<br>
 +
. Miniprep <br>
 +
. Colony PCR for final constructs <br>
 +
 
Line 1,934: Line 2,080:
<br><b>Aug 18</b> <br>
<br><b>Aug 18</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Restriction check for Pbad.TA.dTTs <br>
-
·      <br>
+
. Restriction check for constitutive promoter.CR.GFP.dTT <br>
-
·      <br>
+
. Restriction check for constitutive promoter.RBS.GFP.dTT <br>
-
·   <br>
+
. PCR for CR.GFP.dTT <br>
 +
. PCR for RBS.GFP.dTT <br>
 +
. PCR check for constitutive promoter.CR.GFP.dTT <br>
 +
. PCR check for constitutive promoter.RBS.GFP.dTT <br>
 +
. PCR check for Pbad.TA.dTT <br>
 +
. Restrict check TA.dTTs <br>
 +
. Prepare functional assay for RBS, CRs <br>
 +
. Inoculate candidates <br>
 +
. Prepare pSB3K3 <br>
 +
. Prepare pSB1C3 for oligo ligation <br>
 +
. Prepare I13401 for oligo ligation <br>
 +
. Prepare B0015 for oligo ligation <br>
 +
. Inoue competent cells <br>
 +
. Prepare for Pbad characterization <br>
 +
. Take over the tank B and C arround 1pm-5;30 <br>
 +
 
<br><b>Aug 19</b> <br>
<br><b>Aug 19</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Gel check for O/NPCR products <br>
-
·      <br>
+
. Gel check for PCR products (18/08) <br>
-
·      <br>
+
. miniprep c.Promoter.Lock 1RBS.GFP.dTT <br>
-
·   <br>
+
. miniprep possible candidates <br>
 +
. Restrict TA.dTT <br>
 +
. pSB1C3-I13401 for oligo ligation <br>
 +
. B0015 for oligo ligation <br>
 +
. pSB1C3-J04450 for oligo ligation <br>
 +
. Inoue competent cells <br>
 +
. pBAD-key3-dTT restriction check <br>
 +
 
<br><b>Aug 20</b> <br>
<br><b>Aug 20</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Gel check for Pbad.TA.dTTs <br>
-
·      <br>
+
. purification for B0015, I13401 <br>
-
·      <br>
+
. pSB1C3-J04450 for oligo ligation <br>
-
·   <br>
+
. Gel check for PCR products (19/08) <br>
 +
. PCR check for J23102, LOCK 3 GFP dtt <br>
 +
. restrcition check for key1 dtt <br>
 +
 
<br><b>Aug 21</b> <br>
<br><b>Aug 21</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Colony PCR for final constructs <br>
-
·      <br>
+
. Ligate UST lock <br>
-
·      <br>
+
. restriction check for key1.dTT <br>
-
·   <br>
+
. ligate pbad.key1.dTT <br>
 +
. ligate lock 3.GFP.dTT with C. promoter <br>
 +
. Colony PCR for c. promoter.MCR.GFP.dTT <br>
 +
. competent cells <br>
 +
. Glycerol stocks <br>
 +
. transform HKUSTlock.GFP.dTT <br>
 +
. Transform pSB1C3-HKUSTlock <br>
 +
 
<br><b>Aug 22</b> <br>
<br><b>Aug 22</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. competent cells <br>
-
·      <br>
+
. Colony PCR for pSB1C3-HKUSTlock1 <br>
-
·      <br>
+
. Colony PCR for HKUSTlock1.GFP.dTT <br>
-
·   <br>
+
. miniprep PSB3K3-J04550 for pSB3K3 Backbone <br>
 +
. restrict pSB3K3 for 3 pieces assemblage <br>
 +
. ligate TA-CR into pSB3K3 <br>
 +
. Transform TA-CR <br>
 +
. colony PCR for PBAD-key3-dTT <br>
 +
. inoculate Pbad.key1.dTT <br>
 +
. gel check for C. PCR on 21/08 <br>
 +
. extraction of pSB3K3 <br>
 +
. inoculate HKUSTlock1.GFP.dTT <br>
 +
. inoculate pSB1C3-HKUSTlock1 <br>
 +
 
 +
<br><b>Aug 23</b> <br>
 +
Wet lab<br>
 +
. Gel check for TAs <br>
 +
. colony PCR for UST lock in I13401 and pSB1C3 <br>
 +
. transform ust key <br>
 +
. Check competent cells <br>
 +
 
 +
<br><b>Aug 24</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for Pbad.Key1.dTT <br>
 +
. Gel check for Pbad.Key1.dTT <br>
 +
 
 +
 
       </div>
       </div>
Line 1,970: Line 2,171:
<br><b>Aug 25</b> <br>
<br><b>Aug 25</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Colony PCR for Pbad.Key1.dTT <br>
-
·      <br>
+
. PCR check for CR.GFP.dTT <br>
-
·      <br>
+
. PCR check for Cons. prom.CR.GFP.dTT <br>
-
·   <br>
+
. anneal and ligate ust lock <br>
 +
. anneal and ligate ust key <br>
 +
. transform ust lock1 <br>
 +
. transform ust key <br>
 +
. ligation again key 1 into B0015 <br>
 +
 
<br><b>Aug 26</b> <br>
<br><b>Aug 26</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Colony PCR for "un-named lock" <br>
-
·      <br>
+
. inoculate ones which SEEMS like it contains lock <br>
-
·      <br>
+
. Colony PCR for "un-named key" <br>
-
·   <br>
+
. inoculate ones which SEEMS like it contains key <br>
 +
. colony pcr for Key1.dtt <br>
 +
. Gel check for O/NPCR products <br>
 +
. pcr check for CR.GFP.dTTs <br>
 +
. Inoculate all possible candidates <br>
 +
. Feeling paranoic? Prepare I13401 for CRs <br>
 +
. Streak posible c.p.CR/RBS.GFP.dTT <br>
 +
. make SOC <br>
 +
. single cut check for TAs <br>
 +
. make 5 alpha cell <br>
 +
 
<br><b>Aug 27</b> <br>
<br><b>Aug 27</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. miniprerp candidates <br>
-
·      <br>
+
. pcr check for candidates <br>
-
·      <br>
+
. gel check for o/n pcr <br>
-
·   <br>
+
. restrict check for ust key 1 <br>
 +
. restrick check for key 1 <br>
 +
. Pbad for ust key 1 <br>
 +
. extract ust lock 1 for j23102 <br>
 +
. ligate ust lock and key <br>
 +
. transform ust lock and key <br>
 +
. prepare plates for macroscope <br>
 +
. prepare dna for sequencing <br>
 +
. SOB <br>
 +
. SOC <br>
 +
. oligo annealing <br>
 +
. plasmid PCR for HKUSt lock and key <br>
 +
. overnight digestion of B0034 for control construct <br>
 +
. Digestion of HKUST key-B0015 <br>
 +
 
<br><b>Aug 28</b> <br>
<br><b>Aug 28</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. miniprep locks <br>
-
·      <br>
+
. glycerol stocks for locks <br>
-
·      <br>
+
. minipreps for B0015 and B0034 <br>
-
·   <br>
+
. ligation of pBAD-Hkust key-B0015 <br>
 +
. digestion of HKUST lock <br>
 +
. ligation of HKUST lock with GFP <br>
 +
. ligation of HKUST key and lock (4th) <br>
 +
. ligation of B0034 with GFP <br>
 +
. colony PCR of HKUST key and lock <br>
 +
. inoculation of key and lock <br>
 +
. miniprep <br>
 +
. Key one restrict check <br>
 +
. key one restriction for inserting pBAD <br>
 +
. Restrict and exytact lock3c rbs and ligate into c.promoter <br>
 +
. ligate lock 3c.GFP.dTT with C. promoter <br>
 +
. transform c. promoterlock 3c rbs.gfp.dtt <br>
 +
. Extract mcr/mta <br>
 +
. ligate MCR and MTA into 3K3 <br>
 +
. transform MCR/MTA <br>
 +
. Glycerol stock for locks <br>
 +
. E0240 ligate with C. Promoter <br>
 +
. TB <br>
 +
. parallel ligation and aneling for key 1 <br>
 +
. Sequencing preparations <br>
 +
 
<br><b>Aug 29</b> <br>
<br><b>Aug 29</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. minipreps <br>
-
·      <br>
+
. Gel extraction, purification for O/N digestions <br>
-
·      <br>
+
. competent cells <br>
-
·   <br>
+
. Extract mcr/mta <br>
 +
. ligate MCR and MTA into 3K3 <br>
 +
. transform MCR/MTA <br>
 +
. Glycerol stock for locks <br>
 +
. PCR check for biobrick key1 <br>
 +
 
 +
<br><b>Aug 30</b> <br>
 +
Wet lab<br>
 +
. inoculation of 12 falcons <br>
 +
. Colony PCR for pSB3K3 constructs <br>
 +
. Transform key1 O/N <br>
 +
. miniprep <br>
 +
. Competent cells <br>
 +
. AseI check Key 1 Dtt <br>
 +
 
       </div>
       </div>
     </div>
     </div>
Line 2,021: Line 2,286:
<br><b>Sept 1</b> <br>
<br><b>Sept 1</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. restrict check pBAD- Lost your key-dtt <br>
-
·      <br>
+
. restrict check pBAD-Lost your key-dtt <br>
-
·      <br>
+
. colony PCR for CR.TAs (orthogonality) <br>
-
·   <br>
+
. colony PCR for Key1,dtt <br>
 +
. ligate lock3c.GFP.dTT into c.p plasmid <br>
 +
. transform lock3.gfp.dtt <br>
 +
. Colony PCR for CR.TAs (orthogonality) <br>
 +
. Colony PCR for sherLock-GFP-dtt <br>
 +
. restriciton check for Lost Your Key in PSB1C3 <br>
 +
. transform pbad.lost your key.dtt <br>
 +
. inoculate orthogonality candidates <br>
 +
. Prepara midi-prep for 3k3 <br>
 +
 
<br><b>Sept 2</b> <br>
<br><b>Sept 2</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Gel check for O/N colony pcr for key1 and 3k3 <br>
-
·      <br>
+
. massive prep <br>
-
·      <br>
+
. PCR check sense reverse <br>
-
·   <br>
+
. colony pcr for orthogonality <br>
 +
. colony pcr for cons. pro. lock3crbs.gfp.dtt <br>
 +
. migrate cpromoter E0240 to psb3k3 with mta key <br>
 +
. inoculation of B0034-GFP-dtt <br>
 +
. inoculation of Sherlock-GFP-dtt <br>
 +
. sequencing for sherlock in pSB1C3 <br>
 +
. digestion of B0034-GFP-dtt <br>
 +
. digestion of sherlock-GFP-dtt <br>
 +
 
 +
 
<br><b>Sept 3</b> <br>
<br><b>Sept 3</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. digestion of Pbad MTA dTT <br>
-
·      <br>
+
. digestion of Pbad dTT <br>
-
·      <br>
+
. digestion of J23102 MCR GFP dTT <br>
-
·   <br>
+
. digestion of J23102 E0240(B0032 GFP dTT) <br>
 +
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450) <br>
 +
. Ligation of J23102 MCR GFP dTT with Pbad dTT <br>
 +
. Ligation of J23102 E0240 with Pbad MTA dTT <br>
 +
. Ligation of J23102 E0240 with Pbad dTT <br>
 +
. Plasmid PCR for SherLock-GFP-dtt <br>
 +
. ligation of B0034-GFP-dtt <br>
 +
. ligation of SherLock-GFP-dtt <br>
 +
. sequencing <br>
 +
. check plate for arabinose induced mcr mta while singing <br>
 +
. extraction of key and lock <br>
 +
. restriction check for key with lock <br>
 +
. extration of key1 dtt <br>
 +
. ligation of key1 dtt with pBAD <br>
 +
. Gel check for O/N colonyPCR <br>
 +
. Crazy PCR check <br>
 +
. Check 031 <br>
 +
. ligate lock3c RBS.GFP.dTT into c.p plasmid <br>
 +
. transform lock3c RBS.GFP.dTT <br>
 +
. Midi prep preparations <br>
 +
 
<br><b>Sept 4</b> <br>
<br><b>Sept 4</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Gel purification of digested insert and backbone <br>
-
·      <br>
+
. Ligation of J23102 MCR GFP dTT with Pbad dTT <br>
-
·      <br>
+
. Ligation of J23102 E0240 with Pbad MTA dTT <br>
-
·   <br>
+
. digestion of J23102 E0240(B0032 GFP dTT) <br>
 +
. Transformation of ligated projects mentioned above <br>
 +
. Colony PCR for sherLock-GFP-dtt <br>
 +
. Colony PCR for B0034-GFP-dtt <br>
 +
. Inoculation of successful colonies <br>
 +
. Gel check for O/N Colony PCR <br>
 +
. Gel check for O/N crazy PCR <br>
 +
. Gel check for Erics restriction check <br>
 +
. Inoculation of successful colonies <br>
 +
. Midi-prep preparations <br>
 +
. Gel purification <br>
 +
 
<br><b>Sept 5</b> <br>
<br><b>Sept 5</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. miniprep/glycerol stocck <br>
-
·      <br>
+
. Transform Pbad dTT <br>
-
·      <br>
+
. Transform ligated produce of J23102 MCR GFP dTT with Pbad dTT <br>
-
·   <br>
+
 
Line 2,077: Line 2,391:
<br><b>Sept 10</b> <br>
<br><b>Sept 10</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. colony pcr <br>
-
·      <br>
+
. ligations for final constructs <br>
-
·      <br>
+
. inoculations <br>
-
·   <br>
+
. Pbad.lost your key.dtt <br>
 +
. sherlock <br>
 +
. gel check for colony pcr <br>
 +
. colony pcr <br>
 +
 
<br><b>Sept 11</b> <br>
<br><b>Sept 11</b> <br>
Line 2,091: Line 2,409:
<br><b>Sept 12</b> <br>
<br><b>Sept 12</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. colony pcr <br>
-
·      <br>
+
. restrict key1.dtt for Pbad insertion <br>
-
·      <br>
+
. ligate Pbad with key1.dtt <br>
-
·   <br>
+
. transform key1.dTT <br>
 +
. extract pSB3K3 <br>
 +
. Prepare TB <br>
 +
. Prepare dextrose for SOC <br>
 +
. check sequencing results <br>
 +
. Digestion of CLON041 for constructs <br>
 +
. ligation <br>
 +
. inoculation <br>
 +
. Digestion of CLON 036 <br>
 +
. colony pcr mcr mta <br>
 +
 
       </div>
       </div>
     </div>
     </div>
Line 2,110: Line 2,438:
<br><b>Sept 16</b> <br>
<br><b>Sept 16</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. mini prep Pbad.key1.dtt <br>
-
·      <br>
+
. restrict check pbad.key1.dtt <br>
-
·      <br>
+
. gel check for O/N c.pcr <br>
-
·   <br>
+
. extract pbad.key1 <br>
 +
. inoculate every CR.TA construct <br>
 +
. ligate transform key 1 with locks <br>
 +
. miniprep (B1, B2, and B3) <br>
 +
. plasmid PCR B1, B2 and B3 using sense oligo and VR <br>
 +
. Overnight digestion for ones which give positive results <br>
 +
 
<br><b>Sept 17</b> <br>
<br><b>Sept 17</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. purify pbad.key1 <br>
-
·      <br>
+
. ligate pbad key1 dtt with locks <br>
-
·      <br>
+
. transform locks.Pbad.key1.dtt <br>
-
·   <br>
+
. 2xYT (agar) AMP <br>
 +
. transform all <br>
 +
 
<br><b>Sept 18</b> <br>
<br><b>Sept 18</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Spread a plate <br>
-
·      <br>
+
. miniprep for 3 inoculations <br>
-
·      <br>
+
. Sense oligo and VR PCR for HKUST Lock-dtt construct <br>
-
·   <br>
+
. Overnight digestion of plasmid which gives a band around 1300bp <br>
 +
. EtoHpp for sequencing <br>
 +
. Miniprep construtcs for submission <br>
 +
. innoculation <br>
 +
. transform 031, 034 <br>
 +
. Check "Diagonal constructs" and controls <br>
 +
. migrate CR crontruts into 3K3 <br>
 +
 
<br><b>Sept 19</b> <br>
<br><b>Sept 19</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. miniprep lock.key candidate <br>
-
·      <br>
+
. ligate problematic cr.key <br>
-
·      <br>
+
. restrick check sherlock <br>
-
·   <br>
+
. extrations.... madness <br>
 +
 
 +
<br><b>Sept 20</b> <br>
 +
Wet lab<br>
 +
. miniprep CR.TA <br>
 +
. Colony PCR for CR.TA <br>
 +
. Colony PCR for CR.TA orthogonality <br>
 +
 
 +
<br><b>Sept 21</b> <br>
 +
Wet lab<br>
 +
. miniprep martching CR.TA <br>
 +
. Restriction check by EcoR1 and Pst1 <br>
Line 2,144: Line 2,498:
<br><b>Sept 22</b> <br>
<br><b>Sept 22</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Run Gel check for matching set of CR.TA <br>
-
·      <br>
+
. ligate problrmatic sets <br>
-
·      <br>
+
. transform rpoblematic sets <br>
-
·   <br>
+
. prepare DNA for submission <br>
 +
. Gel purfication for Doyle <br>
 +
. Ligation lock-dtt with cons. promoter <br>
 +
. transformation <br>
 +
. pSB3K3- CRs ligation <br>
 +
 
<br><b>Sept 23</b> <br>
<br><b>Sept 23</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. pSB3K3- CRs transformation <br>
-
·      <br>
+
. Gel extractions <br>
-
·      <br>
+
. Gel purifications <br>
-
·   <br>
+
. TB <br>
 +
. Agar L (+) plates <br>
 +
. Send DNA to BGI for sequencing <br>
 +
. C. PCR? <br>
 +
. Inoculate possible candidates <br>
 +
. ligate problematic sets <br>
 +
. transform confirmed constructs <br>
 +
. inoculation for HKUST lock Construct <br>
 +
. colony PCR For lock <br>
 +
 
<br><b>Sept 24</b> <br>
<br><b>Sept 24</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Competent cells preparations! <br>
-
·      <br>
+
. minipreps <br>
-
·      <br>
+
. colony PCR <br>
-
·   <br>
+
. miniprep for biobrick submission <br>
 +
. send plamsid for sequencing <br>
 +
 
<br><b>Sept 25</b> <br>
<br><b>Sept 25</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. insane prep <br>
-
·      <br>
+
. restriction check <br>
-
·      <br>
+
. Gel check for previous PvuII digestions <br>
-
·   <br>
+
 
<br><b>Sept 26</b> <br>
<br><b>Sept 26</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Gel check PvuII digestions <br>
-
·      <br>
+
. Gel check AseI digestions <br>
-
·      <br>
+
. 3A assembly for non pSB1C3 <br>
-
·   <br>
+
. final batch of ligation for all missing component <br>
 +
. prepare DNA for submission <br>
 +
. Extraction for ligation <br>
 +
. Checking constructs-3 constructs using digestion check <br>
 +
. transformation for LOCK-8 <br>
 +
. Restriction check for controls for MCR/MTA <br>
 +
. Transformation of confirmed plasmid <br>
 +
 
 +
<br><b>Sept 27</b> <br>
 +
Wet lab<br>
 +
. C. PCR for H series... <br>
 +
. inoculate possible candidates <br>
 +
. check self ligated 3K3 <br>
 +
. Gel check for possible candidates AseI <br>
 +
. Gel check for possible candidates PvuII <br>
 +
. Ligated H6 H7 <br>
 +
. transformed H6 H7 <br>
 +
. O/N innoculation of MCR/MTA for characterization <br>
 +
 
 +
<br><b>Sept 28</b> <br>
 +
Wet lab<br>
 +
. Extract pSB1C3 <br>
 +
. Etract 036 <br>
 +
. miniprep "H" series candidates <br>
 +
. Repeat CHL restrictions <br>
 +
. Repeat CHL ligattions <br>
 +
. Repeat CHL transformations <br>
 +
. Gel check for "7" series using NcoI <br>
 +
. Restrict check AseI <br>
 +
. Restrict check NcoI <br>
 +
. Fluorescence measurement of MCR/MTA for characterization. <br>
 +
. Pick a colony dilute and spread MCR/MTA (we don't want bacteria lawn) <br>
 +
 
Line 2,185: Line 2,587:
<br><b>Sept 29</b> <br>
<br><b>Sept 29</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. Restrict check AseI <br>
-
·      <br>
+
. Restrict check NcoI <br>
-
·      <br>
+
. mini prep 6 + MCR/MTA (thanks from doyle) <br>
-
·   <br>
+
. Restriction check for MCR/MTA in pSB3K3. <br>
 +
. restrict 036 <br>
 +
. purify 036 <br>
 +
. ligate monkey with sherlock <br>
 +
. Gel check for NcoI and AseI <br>
 +
. Get pSB1C3 bacbone EcoRI and PstI <br>
 +
. Fluorescence measurement of MCR/MTA for characterization (second trial) <br>
 +
. Innoculate Lock and Keys for crude functional assay. <br>
 +
 
<br><b>Sept 30</b> <br>
<br><b>Sept 30</b> <br>
Wet lab<br>
Wet lab<br>
-
·      <br>
+
. miniprep 11 tubes in shaker c <br>
-
·      <br>
+
. Restrict check H" series with NcoI <br>
-
·      <br>
+
. Restrict inoculated colonies wiht AseI <br>
-
·   <br>
+
. Restrict inoculated colonies wiht NcoI <br>
 +
. Gel purifications <br>
 +
. ligation of H series and some from other series... <br>
 +
. transformtation of 16 confirmed constructs <br>
 +
 
 +
<br><b>Oct 1</b> <br>
 +
Wet lab<br>
 +
. digestion of key3,HKUSTkey? <br>
 +
. miniprep- final constructs <br>
 +
 
       </div>
       </div>

Revision as of 18:37, 15 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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