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Team:Queens Canada/Notebook/Week16
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| + | <tr class="tableizer-firstrow"><th>Date</th><th>Protocol</th><th>People</th><th>DNA (if relevant)</th><th>Quantities and Parameters (if relevant)</th><th>Notes on Protocol</th></tr> <tr><td>08/10/2012</td><td>Fast Digestion</td><td>Victor</td><td>ppFGFP + nFFGFP</td><td> </td><td> </td></tr> <tr><td>08/10/2012</td><td>Gel Extraction</td><td>Victor</td><td>nFFGFP + pFFGFP</td><td> </td><td> </td></tr> <tr><td>08/10/2012</td><td>Digestions</td><td>Kevin</td><td>J48200 w/ pSB1AT3 J13601 Lac operator w/ pSB1A3 746007 Antigen 43 w/ pSB1C3</td><td> </td><td> </td></tr> <tr><td>08/10/2012</td><td>digestion</td><td>Faisal and David</td><td>SmtA and FMT with X and S</td><td> </td><td> </td></tr> <tr><td>08/10/2012</td><td>Gel Extraction</td><td>Phillip</td><td>IPTG (SP), T7 promotor (SP), mCerFC (1,3,4 - 2 trials each)</td><td> </td><td> </td></tr> <tr><td>08/10/2012</td><td>Digestion</td><td>Phillip</td><td>GE IPTG SP [X], GE T7 Prom SP [X]</td><td> </td><td> </td></tr> <tr><td>08/10/2012</td><td>Gel Electrophoresis</td><td>Phillip</td><td> </td><td>1. ladder 2. GE mCerFC 1,2 3. GE mCerFC 1,1 4. GE mCerFC 3,1 5. GE mCerFC 3,2 6. GE mCerFC 4,1 7. GE mCerFC 4,2 8. DIG GE DIG IPTG SP [X] T1 9. DIG GE DIG IPTG SP [X] T2 10. ladder 11. DIG GE DIG T7 Prom SP [X] T1 12. DIG GE DIG T7 Prom SP [X] T1 13. DIG nFFGFP XP 14. DIG nFFGFP 2 XP 15. DIG I15601 (pSB1A3) XP 16. DIG J45200 Banana Odour XP (pSB1AT3) 17. DIG 346002 Antigen 43 XP (pSB1C3) 18. Ladder</td><td> </td></tr> <tr><td>08/10/2012</td><td>Fast Digestion</td><td>Victor</td><td>nFFGFP GE 2, ppFGFP GE 1, MPP Bio-timer k088006, MPP I13601, MPP Bis0A K123000, MPP Antigen 43 K346007</td><td>denatured for 14 mins at 37 degrees. Heat inactivated at 80 degrees for 20 mins</td><td> </td></tr> <tr><td>08/10/2012</td><td>Gel Extraction</td><td>Victor and Kevin</td><td> </td><td>1. ladder 2. nFFGFP DIG XP 3. ppFGFP DIG XP 4. DIG Bio-timer k088006 XP 5. MPP Bis0A K123000 XP T2 6. DIG Antigen 43 K346007 XP 7. DIG KI13601 XP</td><td> </td></tr> <tr><td>08/10/2012</td><td>PCR</td><td>Phillip</td><td>nFFGFP 1+2, ppFGFP 1+2</td><td>25 cycles, 2ul of dna used</td><td>hotstart</td></tr> <tr><td>08/10/2012</td><td>PCR</td><td>Beini</td><td>mCerFC</td><td>25 cycles, 10uL of dna used</td><td>hotstart</td></tr> <tr><td>08/10/2012</td><td>Gel</td><td>Kevin</td><td> </td><td>1. ladder 7. J04500 SP dig 8. nFFGFP dig Xp</td><td>Lanes 2 to 6 are unknown</td></tr> <tr><td>08/10/2012</td><td>Digestion</td><td>Phillip</td><td>J04500 SP</td><td> </td><td> </td></tr> <tr><td>08/10/2012</td><td>Digestion</td><td>Beini</td><td>GE nFFGFP 1 [XP]</td><td> </td><td> </td></tr> <tr><td>08/10/2012</td><td>Gel</td><td>Beini</td><td>1C3 XP GE, 1A3 BisdA XP GE, I13601 1A2 XP GE, J04500 SP GE, K088006 Timer XP GE pSB1A2, GE pFF GFP XP</td><td> </td><td>Small Gel 1</td></tr> <tr><td>08/10/2012</td><td>Gel</td><td>Beini</td><td>mCer FC C, nFF GFP C, T7 prom GE X, IPTG SP GE, DIG GE IPTG SP X, GE T7 prom SP</td><td> </td><td>Small Gel 2</td></tr> <tr><td>08/10//2012</td><td>Gel extraction</td><td>Beini, Kevin</td><td>mCer FC, pFF GFP, nFF GFP GE XP, pFF GFP XS</td><td>loaded 4 uL loading dye : 16 uL DNA; for digests with FD green, loaded all 30 uL</td><td> </td></tr> <tr><td>08/11//2012</td><td>Liquid cultures</td><td>Kevin</td><td>K314101 (low exp cassette), K314100 (high conc exp), K314103 (lac ind cassette)</td><td>3.68 uL [C] added to each</td><td> </td></tr> <tr><td>08/11//2012</td><td>Miniprep</td><td>Kevin</td><td>K081005 (cons. prom. + RBS) x 2, K314104 (T7 exp. cassette) x 2</td><td> </td><td> </td></tr> <tr><td>08/11//2012</td><td>Digestions</td><td>Kevin</td><td>MPPs: K081005, K314104 with SP, GEs: nFF GFP, pFF GFP with XP</td><td>MPPs: 5 uL DNA, 20 uL total rxn volume, GEs: 15 uL DNA, 30 uL total rxn volume </td><td>Fast Digest</td></tr> <tr><td>12/08/2012</td><td>PCR</td><td>Beini</td><td>GE pFliC1 + GFP + FliC2</td><td> </td><td> </td></tr> <tr><td>12/08/2012</td><td>Digestion with XS</td><td>Faisal</td><td>PCR products SmtA and fMt</td><td> </td><td>Fast Digest, enzymatic purification using Biobasic kit</td></tr> <tr><td>12/08/2012</td><td>PCR overlap extension of MBPs with FliC1</td><td>Faisal</td><td>SmtA, fMt, FliC1</td><td> </td><td> </td></tr> <tr><td>12/08/2012</td><td>Soft agar (0.25%) plates</td><td>Beini</td><td> </td><td>made 7 plates each of [C], [amp], [tet]</td><td> </td></tr> <tr><td>12/08/2012</td><td>Miniprep</td><td>Beini</td><td>K314100 high exp cassette, K314101 low exp cassette, K314103 lac expression cassette</td><td> </td><td> </td></tr> <tr><td>12/08/2012</td><td>Gel</td><td>Beini</td><td>K081005 SP GE, K314104 SP GE</td><td> </td><td> </td></tr> <tr><td>12/08/2012</td><td>Gel</td><td>Beini</td><td>nFF GFP GE, nFF GFP XP GE, pFF GFP XP GE, MPP K314100, MPP K314101, MPP K314103</td><td> </td><td> </td></tr> <tr><td>12/08/2012</td><td>Gel extraction</td><td>Kevin</td><td>nFF GFP, nFF GFP XP, pFF GFP XP, K081005 SP, K314104 SP</td><td> </td><td> </td></tr> <tr><td>12/08/2012</td><td>Gel</td><td>Kevin</td><td>MPP K081005, MPP K13104, pFF GFP, nFF GFP, pFF GFP XS</td><td> </td><td> </td></tr> <tr><td>12/08/2012</td><td>Digestions</td><td>Beini</td><td>MPP K314100 with SP, MPP K314103 with SP, GE nFF GFP with XP</td><td>digested MPPs for 12 mins, GEs for 30 mins</td><td> </td></tr> <tr><td>12/08/2012</td><td>Ligations</td><td>Kevin</td><td>pFF GFP in 1A2 from K088006</td><td>used 6 and 8 uL of vector for 1:1 and 3:1 ligations</td><td> </td></tr> <tr><td>12/08/2012</td><td>Gel</td><td>Kevin</td><td>1:1 lig, 3:1 lig, DIG XP nFF GFP, DIG SP K314100, DIG SP K314103</td><td> </td><td>gel extracted the digestions</td></tr> <tr><td>12/08/2012</td><td>Heat Shock Transformation using Motility Plates</td><td>Kevin</td><td> </td><td> </td><td> </td></tr> <tr><td>13/08/2012</td><td>Ligations</td><td>Victor</td><td>nFF GFP XP GE + 1A3 XP GE. nFF GFP XP GE + 1A3 XP GE, nFF GFP XP GE + T7 GE SP, nFF GFP XP GE + K314100 GE SP, nFF GFP ctrl</td><td> </td><td> </td></tr> <tr><td>13/08/2012</td><td>Gel</td><td>David, Kevin</td><td>fMT FliC, SmA FliC, DIG nFF GFP XP, LIG nFF GFP XP 1A3, LIG nFF GFP XP T7, LIG nFF GFP XP K314100</td><td> </td><td> </td></tr> <tr><td>13/08/2012</td><td>Digestions</td><td>Victor</td><td>nFF GFP GE (3) with XP</td><td> </td><td> </td></tr> <tr><td>13/08/2012</td><td>Gel extraction</td><td>David</td><td>fMt FliC, SmtA FliC from above gel</td><td> </td><td> </td></tr> <tr><td>13/08/2012</td><td>Gel</td><td>Kevin</td><td>nFF GFP XP, K314100 SP, SmtA FliC1, fMt FliC1</td><td> </td><td> </td></tr> <tr><td>13/08/2012</td><td>Ligations</td><td>Kevin</td><td>Vectors: K314104, K081005, K314100, K314003, J04500, Insert: nFF GFP XP</td><td>1:1, 2:1 insert:vector ratios</td><td>plated 100 uL on [K], 100 uL on [AK], spun down the rest at 12000 rpm and resuspended 100 uL on [AK]</td></tr> <tr><td>13/08/2012</td><td>Digestions</td><td>Beini</td><td>MPP J33204 xylE + RBS with XP, nFF GFP GE with XP</td><td>digested MPP for 13 mins at 37C, digested GEs for 30 mins at 37C</td><td>iced while waiting for 80C bath to heat up</td></tr> <tr><td>14/08/2012</td><td>Liquid cultures</td><td>Kevin</td><td>pFF GFP 1A3 1:3 lig (the rest)</td><td> </td><td> </td></tr> <tr><td>14/08/2012</td><td>Digestions</td><td>David</td><td>GEs SmtA + FliC, fMt + FliC1 with SpeI</td><td> </td><td> </td></tr> <tr><td>14/08/2012</td><td>Digestions</td><td>Victor</td><td>nFF GFP GE with XP</td><td> </td><td> </td></tr> <tr><td>14/08/2012</td><td>Digestions of linearized plasmid backbone</td><td>Victor</td><td>pSB1C3, pSB1A3 with XP and DpnI</td><td> </td><td>digestion purification</td></tr> <tr><td>14/08/2012</td><td>Gel Extraction</td><td>Victor</td><td>DIG XP nFF GFP, npp pFF GFP 1A3, DIG EP pFF GFP 1A3, pSB1C3 XP</td><td> </td><td>did enzymatic purification of pSB1C3 digest instead of gel purification</td></tr> <tr><td>14/08/2012</td><td>Gel Extraction</td><td>Kevin</td><td>nFF GFP XP, nFF GFP XP, DIG xylE + RBS XP</td><td> </td><td> </td></tr> <tr><td>14/08/2012</td><td>Miniprep</td><td>Kevin</td><td>LIG pFF GFP + 1A3 1:3 (the rest)</td><td> </td><td> </td></tr> <tr><td>14/08/2012</td><td>Digestion</td><td>Kevin</td><td>LIG pFF GFP + 1A3 1:3 (the rest) with EP</td><td> </td><td> </td></tr> <tr><td>14/08/2012</td><td>PCR overlap extension</td><td>Faisal</td><td>fliC1 + MBP, fliC2</td><td>did 1:1, 2:1, 3:1 ratios of fliC1 + MBP : fliC2</td><td>digestion purification of SmtA + fliC1 and fMT + fliC1 digested with SpeI</td></tr> <tr><td>14/08/2012</td><td>Ligations</td><td>Victor, Mitangi</td><td>pSB1A3 + nFF GFP</td><td>did 1:1, 2:1 ratios of insert : vector</td><td> </td></tr> <tr><td>14/08/2012</td><td>Gel to check for concentrations</td><td>Victor</td><td>nFF GFP GE XP, nFF GFP GE XP, GE xylE + RBS XP plasmid, GE xylE + RBS</td><td> </td><td> </td></tr> <tr><td>14/08/2012</td><td>Liquid cultures of fluorescent colonies</td><td>Kevin, Faisal</td><td>nFF GFP + J04500 (6 trials + 3 ctrls)</td><td># of fluorescent colonies , AK 1:1 100 uL - 10,AK 1:1 the rest - 21, K 1:1 100 uL - 50, AK: 1:3 100 uL - 0, AK 1:3 the rest - 2, K 1:3 100 uL - 2</td><td> </td></tr> <tr><td>14/08/2012</td><td>Heat shock transformation</td><td>Beini</td><td>nFF GFP + 1A3</td><td>1:1, 2:1, ctrl</td><td> </td></tr> <tr><td>14/08/2012</td><td>Gel extraction</td><td>Faisal, David</td><td>fmt FliC1 + FliC2, SmtA FliC1 + FliC2</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>Minipreps</td><td>Kevin</td><td>nFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>PCR of Full FliC MBP Gel Extractions</td><td>David, Faisal</td><td>fmt FliC1 + FliC2, SmtA FliC1 + FliC2</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>Digestion</td><td>Phillip</td><td>nFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest with ES</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>Gel</td><td>Victor</td><td>DIG B0015 EX, DIG B0017 EX. DIG nFF GFP + J04500 ES, pFF GFP + 1A3, MPP nFF GFP + J04506, MPP nFF GFP + J04560</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>Ligations</td><td>Victor</td><td>GE nFF GFP XP, 1C3 XP GE</td><td>did 1:1, 2:1 insert:vector ratios</td><td> </td></tr> <tr><td>15/08/2012</td><td>Gel</td><td>Phillip</td><td>MPP nFF + J04500, MPP pFF GFP 1A3</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>Liquid culture</td><td>Phillip</td><td>nFF GFP + 1A3</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>Streak plating</td><td>Victor</td><td>nFF GFP + J04500</td><td>1:2, 1:3, 1:1</td><td> </td></tr> <tr><td>15/08/2012</td><td>Gel Extraction of FF MBP products from PCR overlap extension</td><td>Faisal</td><td>SmtA FliC1 + FliC2</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>Digestioin</td><td>Victor</td><td>B0015, B0017 terminators with EX</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>PCR overlap extension</td><td>Kevin</td><td>mCer DP XS, nFF GFP + J04500</td><td>need a super high insert:vector ratio, >>50:1 extension time 5 min 30 s, 17 cycles</td><td> </td></tr> <tr><td>15/08/2012</td><td>Liquid Culture</td><td>Kevin</td><td>nFFGFP 1A3 4 trials, J04500</td><td> </td><td> </td></tr> <tr><td>15/08/2012</td><td>Motility plate inoculation</td><td>Kevin</td><td>nFF GFP + J04500 6 trials</td><td>3 uL stab + inject per spot</td><td>1 stop only on pipette</td></tr> <tr><td>15/08/2012</td><td>Digestion of mCer OE</td><td>Kevin</td><td>mCer OE</td><td>17 uL DNA, 1 uL DpnI, 2 uL buffer Tango</td><td> </td></tr> <tr><td>16/08/2012</td><td>Digestion of PP mCer and PP mCit with XS</td><td>David</td><td>PP mCer, PP mCit</td><td> </td><td>ran out of SpeI, only got a bit of S for mCit1; mCer2 and mCit2 with X only</td></tr> <tr><td>16/08/2012</td><td>Ligations</td><td>Victor</td><td>GE nFF GFP XP + 1C3 XP GE from Antigen-43</td><td>1:1 insert:vector</td><td> </td></tr> <tr><td>16/08/2012</td><td>Digestion</td><td>Victor</td><td>Luciferase PCR products with XS</td><td> </td><td> </td></tr> <tr><td>16/08/2012</td><td>Miniprep</td><td>Kevin</td><td>nFF GFP + 1A3, J04500, nFF GFP + 1A3, nFF GFP + 1A3</td><td> </td><td> </td></tr> <tr><td>16/08/2012</td><td>Digestion with EP</td><td>Kevin</td><td>nFF GFP + 1A3, nFF GFP + 1A3, nFF GFP + 1A3</td><td>digested 10 uL of dna</td><td>NEB</td></tr> <tr><td>16/08/2012</td><td>Gel extraction</td><td>Victor, Beini</td><td>DIG mCer XS, DIG mCer X, dig mCit XS, DIG mCit X, DIG luciferase XS, gelled to check concs: GE nFF GFP XP, GE xylE + RBS XP plasmid, GE xylE + RBS XP</td><td> </td><td>DIG mCit XS did not show up on gel</td></tr> <tr><td>16/08/2012</td><td>Gel</td><td>Kevin</td><td>MPP nff GFP 2:1 the rest, DIG EP nFF GFP 2:1 the rest, MPP J04500, MPP nFF GFP 1A3, DIG EP nFF GFP 1A3</td><td> </td><td> </td></tr> <tr><td>16/08/2012</td><td>Digestion w/ EcoRI-HF</td><td>Kevin</td><td>MPP nFF GFP 1A3 2:1 the rest</td><td> </td><td> </td></tr> <tr><td>16/08/2012</td><td>Gel</td><td>Kevin</td><td>GE mCer X, GE luciferase XS, DIG nFF GFP E</td><td> </td><td> </td></tr> <tr><td>16/08/2012</td><td>PCR overlap extension</td><td>Kevin</td><td>inserts: mCer, luciferase, vector: nFF GFP + J04500 K</td><td>8 mins. 30 s extension time, 18 cycles</td><td>1 trial with Kapa, 1 trial with Phusion</td></tr> <tr><td>16/08/2012</td><td>Liquid cultures</td><td>Kevin</td><td>fluorescent colony AK nFF GFP J04500 1:3 the rest from 08/14 streak plate</td><td> </td><td> </td></tr> <tr><td>16/08/2012</td><td>Spectrometer</td><td>Kevin</td><td> </td><td>OD600, 2XTY against no reference: 0.055, J04500 culture: 1.878, nFF GFP + J04500 1:2 K: 1.701 (using a different cuvette = 1.731),nFF GFP + J04500 1:2 AK: 1.760</td><td> </td></tr> <tr><td>16/08/2012</td><td>Motility plate inoculations</td><td>Kevin</td><td>J04500, nFF GFP + J04500 1:2 K, nFF GFP + J04500 1:2 AK</td><td>only J04500 shows motility</td><td> </td></tr> <tr><td>16/08/2012</td><td>Gel</td><td>Phillip</td><td>PCR-OE products: luciferase Kapa, luciferase Phusion</td><td> </td><td> </td></tr> <tr><td>17/08/2012</td><td>250 mL inoculations of 2XTY</td><td>Victor</td><td>AK nFF GFP + J04500 1:2, AK J04500</td><td> </td><td> </td></tr> <tr><td>17/08/2012</td><td>Digestions with DpnI using Tango buffer</td><td>Phillip</td><td>PCR-OE products: luciferase Kapa, luciferase Phusion</td><td> </td><td> </td></tr> <tr><td>17/08/2012</td><td>Using the ultracentrifuge to obtain a bacterial cell pellet</td><td>Victor, as told by Jun</td><td> </td><td> </td><td>use JA-10 rotor and 250 mL bottles 10 000 rpm, 6000 speed, 10 mins., 4C</td></tr> <tr><td>17/08/2012</td><td>Tris-HCl buffer</td><td>Victor</td><td> </td><td>12.1 g Tris, 800 mL H2O, 7.7 mL HCl until pH 7.8 is reached, fill the rest to 1 L of solution, autoclave at Liquid 1 setting</td><td> </td></tr> <tr><td>17/08/2012</td><td>Liquid culture of ligations</td><td>Beini</td><td>nFF GFP 1C3 100 pellet, nFF GFP 1C3 100 uL, nFF GFP 1A2 1:1 pellet, nFF GFP 1A2 1:1 100 uL</td><td> </td><td> </td></tr> <tr><td>18/08/2012</td><td>Isolation of flagella</td><td>Beini</td><td>AK nFF GFP + J04500 1:2, AK J04500</td><td> </td><td>resuspended cell pellet in 100 mL Tris-HCl, blended 1 min 30 s in Waring blender at 4C, centrifuged 10 mins. at 4C, 10 000 rpm, speed 6000 , centrifuged supernatant for 1 h at 4C, 20 000 rpm, speed 6000</td></tr> <tr><td>18/08/2012</td><td>Liquid cultures</td><td>Mitangi</td><td>AK DIG luciferase Kapa DpnI</td><td> </td><td> </td></tr> <tr><td>18/08/2012</td><td>Miniprep</td><td>Mitangi</td><td>nFF GFP 1A2, nFF GFP 1C3</td><td> </td><td> </td></tr> <tr><td>18/08/2012</td><td>Digestion with PstI</td><td>Mitangi</td><td>nFF GFP 1A2, nFF GFP 1C3, nFF GFP 1A3 (all MPPs)</td><td> </td><td> </td></tr> <tr><td>18/08/2012</td><td>Gel</td><td>Beini</td><td>MPP nFF GFP 1A3, DIG P nFF GFP 1A3, MPP nFF GFP 1A2, DIG nFF GFP 1A2 P, MPP nFF GFP 1A2, DIG P nFF GFP 1C3, DIG P nFF GFO 1C3</td><td> </td><td> </td></tr> <tr><td>18/08/2012</td><td>Miniprep</td><td>Beini</td><td>AK nFF GFP J04500 1:3 the rest</td><td> </td><td>made glycerol stock</td></tr> <tr><td>18/08/2012</td><td>Miniprep</td><td>Beini</td><td>AK DIG luciferase Kapa DpnI (PCR OE of luciferase + nFF GFP J04500)</td><td> </td><td></td></tr></table> | ||
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Latest revision as of 22:38, 26 October 2012
Control
Notebook - Week 16
| Date | Protocol | People | DNA (if relevant) | Quantities and Parameters (if relevant) | Notes on Protocol |
|---|---|---|---|---|---|
| 08/10/2012 | Fast Digestion | Victor | ppFGFP + nFFGFP | ||
| 08/10/2012 | Gel Extraction | Victor | nFFGFP + pFFGFP | ||
| 08/10/2012 | Digestions | Kevin | J48200 w/ pSB1AT3 J13601 Lac operator w/ pSB1A3 746007 Antigen 43 w/ pSB1C3 | ||
| 08/10/2012 | digestion | Faisal and David | SmtA and FMT with X and S | ||
| 08/10/2012 | Gel Extraction | Phillip | IPTG (SP), T7 promotor (SP), mCerFC (1,3,4 - 2 trials each) | ||
| 08/10/2012 | Digestion | Phillip | GE IPTG SP [X], GE T7 Prom SP [X] | ||
| 08/10/2012 | Gel Electrophoresis | Phillip | 1. ladder 2. GE mCerFC 1,2 3. GE mCerFC 1,1 4. GE mCerFC 3,1 5. GE mCerFC 3,2 6. GE mCerFC 4,1 7. GE mCerFC 4,2 8. DIG GE DIG IPTG SP [X] T1 9. DIG GE DIG IPTG SP [X] T2 10. ladder 11. DIG GE DIG T7 Prom SP [X] T1 12. DIG GE DIG T7 Prom SP [X] T1 13. DIG nFFGFP XP 14. DIG nFFGFP 2 XP 15. DIG I15601 (pSB1A3) XP 16. DIG J45200 Banana Odour XP (pSB1AT3) 17. DIG 346002 Antigen 43 XP (pSB1C3) 18. Ladder | ||
| 08/10/2012 | Fast Digestion | Victor | nFFGFP GE 2, ppFGFP GE 1, MPP Bio-timer k088006, MPP I13601, MPP Bis0A K123000, MPP Antigen 43 K346007 | denatured for 14 mins at 37 degrees. Heat inactivated at 80 degrees for 20 mins | |
| 08/10/2012 | Gel Extraction | Victor and Kevin | 1. ladder 2. nFFGFP DIG XP 3. ppFGFP DIG XP 4. DIG Bio-timer k088006 XP 5. MPP Bis0A K123000 XP T2 6. DIG Antigen 43 K346007 XP 7. DIG KI13601 XP | ||
| 08/10/2012 | PCR | Phillip | nFFGFP 1+2, ppFGFP 1+2 | 25 cycles, 2ul of dna used | hotstart |
| 08/10/2012 | PCR | Beini | mCerFC | 25 cycles, 10uL of dna used | hotstart |
| 08/10/2012 | Gel | Kevin | 1. ladder 7. J04500 SP dig 8. nFFGFP dig Xp | Lanes 2 to 6 are unknown | |
| 08/10/2012 | Digestion | Phillip | J04500 SP | ||
| 08/10/2012 | Digestion | Beini | GE nFFGFP 1 [XP] | ||
| 08/10/2012 | Gel | Beini | 1C3 XP GE, 1A3 BisdA XP GE, I13601 1A2 XP GE, J04500 SP GE, K088006 Timer XP GE pSB1A2, GE pFF GFP XP | Small Gel 1 | |
| 08/10/2012 | Gel | Beini | mCer FC C, nFF GFP C, T7 prom GE X, IPTG SP GE, DIG GE IPTG SP X, GE T7 prom SP | Small Gel 2 | |
| 08/10//2012 | Gel extraction | Beini, Kevin | mCer FC, pFF GFP, nFF GFP GE XP, pFF GFP XS | loaded 4 uL loading dye : 16 uL DNA; for digests with FD green, loaded all 30 uL | |
| 08/11//2012 | Liquid cultures | Kevin | K314101 (low exp cassette), K314100 (high conc exp), K314103 (lac ind cassette) | 3.68 uL [C] added to each | |
| 08/11//2012 | Miniprep | Kevin | K081005 (cons. prom. + RBS) x 2, K314104 (T7 exp. cassette) x 2 | ||
| 08/11//2012 | Digestions | Kevin | MPPs: K081005, K314104 with SP, GEs: nFF GFP, pFF GFP with XP | MPPs: 5 uL DNA, 20 uL total rxn volume, GEs: 15 uL DNA, 30 uL total rxn volume | Fast Digest |
| 12/08/2012 | PCR | Beini | GE pFliC1 + GFP + FliC2 | ||
| 12/08/2012 | Digestion with XS | Faisal | PCR products SmtA and fMt | Fast Digest, enzymatic purification using Biobasic kit | |
| 12/08/2012 | PCR overlap extension of MBPs with FliC1 | Faisal | SmtA, fMt, FliC1 | ||
| 12/08/2012 | Soft agar (0.25%) plates | Beini | made 7 plates each of [C], [amp], [tet] | ||
| 12/08/2012 | Miniprep | Beini | K314100 high exp cassette, K314101 low exp cassette, K314103 lac expression cassette | ||
| 12/08/2012 | Gel | Beini | K081005 SP GE, K314104 SP GE | ||
| 12/08/2012 | Gel | Beini | nFF GFP GE, nFF GFP XP GE, pFF GFP XP GE, MPP K314100, MPP K314101, MPP K314103 | ||
| 12/08/2012 | Gel extraction | Kevin | nFF GFP, nFF GFP XP, pFF GFP XP, K081005 SP, K314104 SP | ||
| 12/08/2012 | Gel | Kevin | MPP K081005, MPP K13104, pFF GFP, nFF GFP, pFF GFP XS | ||
| 12/08/2012 | Digestions | Beini | MPP K314100 with SP, MPP K314103 with SP, GE nFF GFP with XP | digested MPPs for 12 mins, GEs for 30 mins | |
| 12/08/2012 | Ligations | Kevin | pFF GFP in 1A2 from K088006 | used 6 and 8 uL of vector for 1:1 and 3:1 ligations | |
| 12/08/2012 | Gel | Kevin | 1:1 lig, 3:1 lig, DIG XP nFF GFP, DIG SP K314100, DIG SP K314103 | gel extracted the digestions | |
| 12/08/2012 | Heat Shock Transformation using Motility Plates | Kevin | |||
| 13/08/2012 | Ligations | Victor | nFF GFP XP GE + 1A3 XP GE. nFF GFP XP GE + 1A3 XP GE, nFF GFP XP GE + T7 GE SP, nFF GFP XP GE + K314100 GE SP, nFF GFP ctrl | ||
| 13/08/2012 | Gel | David, Kevin | fMT FliC, SmA FliC, DIG nFF GFP XP, LIG nFF GFP XP 1A3, LIG nFF GFP XP T7, LIG nFF GFP XP K314100 | ||
| 13/08/2012 | Digestions | Victor | nFF GFP GE (3) with XP | ||
| 13/08/2012 | Gel extraction | David | fMt FliC, SmtA FliC from above gel | ||
| 13/08/2012 | Gel | Kevin | nFF GFP XP, K314100 SP, SmtA FliC1, fMt FliC1 | ||
| 13/08/2012 | Ligations | Kevin | Vectors: K314104, K081005, K314100, K314003, J04500, Insert: nFF GFP XP | 1:1, 2:1 insert:vector ratios | plated 100 uL on [K], 100 uL on [AK], spun down the rest at 12000 rpm and resuspended 100 uL on [AK] |
| 13/08/2012 | Digestions | Beini | MPP J33204 xylE + RBS with XP, nFF GFP GE with XP | digested MPP for 13 mins at 37C, digested GEs for 30 mins at 37C | iced while waiting for 80C bath to heat up |
| 14/08/2012 | Liquid cultures | Kevin | pFF GFP 1A3 1:3 lig (the rest) | ||
| 14/08/2012 | Digestions | David | GEs SmtA + FliC, fMt + FliC1 with SpeI | ||
| 14/08/2012 | Digestions | Victor | nFF GFP GE with XP | ||
| 14/08/2012 | Digestions of linearized plasmid backbone | Victor | pSB1C3, pSB1A3 with XP and DpnI | digestion purification | |
| 14/08/2012 | Gel Extraction | Victor | DIG XP nFF GFP, npp pFF GFP 1A3, DIG EP pFF GFP 1A3, pSB1C3 XP | did enzymatic purification of pSB1C3 digest instead of gel purification | |
| 14/08/2012 | Gel Extraction | Kevin | nFF GFP XP, nFF GFP XP, DIG xylE + RBS XP | ||
| 14/08/2012 | Miniprep | Kevin | LIG pFF GFP + 1A3 1:3 (the rest) | ||
| 14/08/2012 | Digestion | Kevin | LIG pFF GFP + 1A3 1:3 (the rest) with EP | ||
| 14/08/2012 | PCR overlap extension | Faisal | fliC1 + MBP, fliC2 | did 1:1, 2:1, 3:1 ratios of fliC1 + MBP : fliC2 | digestion purification of SmtA + fliC1 and fMT + fliC1 digested with SpeI |
| 14/08/2012 | Ligations | Victor, Mitangi | pSB1A3 + nFF GFP | did 1:1, 2:1 ratios of insert : vector | |
| 14/08/2012 | Gel to check for concentrations | Victor | nFF GFP GE XP, nFF GFP GE XP, GE xylE + RBS XP plasmid, GE xylE + RBS | ||
| 14/08/2012 | Liquid cultures of fluorescent colonies | Kevin, Faisal | nFF GFP + J04500 (6 trials + 3 ctrls) | # of fluorescent colonies , AK 1:1 100 uL - 10,AK 1:1 the rest - 21, K 1:1 100 uL - 50, AK: 1:3 100 uL - 0, AK 1:3 the rest - 2, K 1:3 100 uL - 2 | |
| 14/08/2012 | Heat shock transformation | Beini | nFF GFP + 1A3 | 1:1, 2:1, ctrl | |
| 14/08/2012 | Gel extraction | Faisal, David | fmt FliC1 + FliC2, SmtA FliC1 + FliC2 | ||
| 15/08/2012 | Minipreps | Kevin | nFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest | ||
| 15/08/2012 | PCR of Full FliC MBP Gel Extractions | David, Faisal | fmt FliC1 + FliC2, SmtA FliC1 + FliC2 | ||
| 15/08/2012 | Digestion | Phillip | nFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest with ES | ||
| 15/08/2012 | Gel | Victor | DIG B0015 EX, DIG B0017 EX. DIG nFF GFP + J04500 ES, pFF GFP + 1A3, MPP nFF GFP + J04506, MPP nFF GFP + J04560 | ||
| 15/08/2012 | Ligations | Victor | GE nFF GFP XP, 1C3 XP GE | did 1:1, 2:1 insert:vector ratios | |
| 15/08/2012 | Gel | Phillip | MPP nFF + J04500, MPP pFF GFP 1A3 | ||
| 15/08/2012 | Liquid culture | Phillip | nFF GFP + 1A3 | ||
| 15/08/2012 | Streak plating | Victor | nFF GFP + J04500 | 1:2, 1:3, 1:1 | |
| 15/08/2012 | Gel Extraction of FF MBP products from PCR overlap extension | Faisal | SmtA FliC1 + FliC2 | ||
| 15/08/2012 | Digestioin | Victor | B0015, B0017 terminators with EX | ||
| 15/08/2012 | PCR overlap extension | Kevin | mCer DP XS, nFF GFP + J04500 | need a super high insert:vector ratio, >>50:1 extension time 5 min 30 s, 17 cycles | |
| 15/08/2012 | Liquid Culture | Kevin | nFFGFP 1A3 4 trials, J04500 | ||
| 15/08/2012 | Motility plate inoculation | Kevin | nFF GFP + J04500 6 trials | 3 uL stab + inject per spot | 1 stop only on pipette |
| 15/08/2012 | Digestion of mCer OE | Kevin | mCer OE | 17 uL DNA, 1 uL DpnI, 2 uL buffer Tango | |
| 16/08/2012 | Digestion of PP mCer and PP mCit with XS | David | PP mCer, PP mCit | ran out of SpeI, only got a bit of S for mCit1; mCer2 and mCit2 with X only | |
| 16/08/2012 | Ligations | Victor | GE nFF GFP XP + 1C3 XP GE from Antigen-43 | 1:1 insert:vector | |
| 16/08/2012 | Digestion | Victor | Luciferase PCR products with XS | ||
| 16/08/2012 | Miniprep | Kevin | nFF GFP + 1A3, J04500, nFF GFP + 1A3, nFF GFP + 1A3 | ||
| 16/08/2012 | Digestion with EP | Kevin | nFF GFP + 1A3, nFF GFP + 1A3, nFF GFP + 1A3 | digested 10 uL of dna | NEB |
| 16/08/2012 | Gel extraction | Victor, Beini | DIG mCer XS, DIG mCer X, dig mCit XS, DIG mCit X, DIG luciferase XS, gelled to check concs: GE nFF GFP XP, GE xylE + RBS XP plasmid, GE xylE + RBS XP | DIG mCit XS did not show up on gel | |
| 16/08/2012 | Gel | Kevin | MPP nff GFP 2:1 the rest, DIG EP nFF GFP 2:1 the rest, MPP J04500, MPP nFF GFP 1A3, DIG EP nFF GFP 1A3 | ||
| 16/08/2012 | Digestion w/ EcoRI-HF | Kevin | MPP nFF GFP 1A3 2:1 the rest | ||
| 16/08/2012 | Gel | Kevin | GE mCer X, GE luciferase XS, DIG nFF GFP E | ||
| 16/08/2012 | PCR overlap extension | Kevin | inserts: mCer, luciferase, vector: nFF GFP + J04500 K | 8 mins. 30 s extension time, 18 cycles | 1 trial with Kapa, 1 trial with Phusion |
| 16/08/2012 | Liquid cultures | Kevin | fluorescent colony AK nFF GFP J04500 1:3 the rest from 08/14 streak plate | ||
| 16/08/2012 | Spectrometer | Kevin | OD600, 2XTY against no reference: 0.055, J04500 culture: 1.878, nFF GFP + J04500 1:2 K: 1.701 (using a different cuvette = 1.731),nFF GFP + J04500 1:2 AK: 1.760 | ||
| 16/08/2012 | Motility plate inoculations | Kevin | J04500, nFF GFP + J04500 1:2 K, nFF GFP + J04500 1:2 AK | only J04500 shows motility | |
| 16/08/2012 | Gel | Phillip | PCR-OE products: luciferase Kapa, luciferase Phusion | ||
| 17/08/2012 | 250 mL inoculations of 2XTY | Victor | AK nFF GFP + J04500 1:2, AK J04500 | ||
| 17/08/2012 | Digestions with DpnI using Tango buffer | Phillip | PCR-OE products: luciferase Kapa, luciferase Phusion | ||
| 17/08/2012 | Using the ultracentrifuge to obtain a bacterial cell pellet | Victor, as told by Jun | use JA-10 rotor and 250 mL bottles 10 000 rpm, 6000 speed, 10 mins., 4C | ||
| 17/08/2012 | Tris-HCl buffer | Victor | 12.1 g Tris, 800 mL H2O, 7.7 mL HCl until pH 7.8 is reached, fill the rest to 1 L of solution, autoclave at Liquid 1 setting | ||
| 17/08/2012 | Liquid culture of ligations | Beini | nFF GFP 1C3 100 pellet, nFF GFP 1C3 100 uL, nFF GFP 1A2 1:1 pellet, nFF GFP 1A2 1:1 100 uL | ||
| 18/08/2012 | Isolation of flagella | Beini | AK nFF GFP + J04500 1:2, AK J04500 | resuspended cell pellet in 100 mL Tris-HCl, blended 1 min 30 s in Waring blender at 4C, centrifuged 10 mins. at 4C, 10 000 rpm, speed 6000 , centrifuged supernatant for 1 h at 4C, 20 000 rpm, speed 6000 | |
| 18/08/2012 | Liquid cultures | Mitangi | AK DIG luciferase Kapa DpnI | ||
| 18/08/2012 | Miniprep | Mitangi | nFF GFP 1A2, nFF GFP 1C3 | ||
| 18/08/2012 | Digestion with PstI | Mitangi | nFF GFP 1A2, nFF GFP 1C3, nFF GFP 1A3 (all MPPs) | ||
| 18/08/2012 | Gel | Beini | MPP nFF GFP 1A3, DIG P nFF GFP 1A3, MPP nFF GFP 1A2, DIG nFF GFP 1A2 P, MPP nFF GFP 1A2, DIG P nFF GFP 1C3, DIG P nFF GFO 1C3 | ||
| 18/08/2012 | Miniprep | Beini | AK nFF GFP J04500 1:3 the rest | made glycerol stock | |
| 18/08/2012 | Miniprep | Beini | AK DIG luciferase Kapa DpnI (PCR OE of luciferase + nFF GFP J04500) |
Given that the vast majority of our work now is in the lab, we will only be updating the "Labwork" section of the Notebook.
