Team:Queens Canada/Notebook/Week16

From 2012.igem.org

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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>12-08-2012</td><td>PCR</td><td>Beini</td><td>GE pFliC1 + GFP + FliC2</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>12-08-2012</td><td>Digestion with XS</td><td>Faisal </td><td>PCR products SmtA and fMt</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>12-08-2012</td><td>Soft agar (0.25%) plates </td><td>Beini</td><td>&nbsp;</td><td>made 7 plates each of [C], [amp], [tet]</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>12-08-2012</td><td>Gel</td><td>Beini</td><td>K081005 SP GE, K314104 SP GE</td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>12-08-2012</td><td>Digestions </td><td>Beini</td><td>"MPP K314100 with SP</td></tr> <tr><td>MPP K314103 with SP</td></tr> <tr><td>GE nFF GFP  with XP"</td><td>digested MPPs for 12 mins, GEs for 30 mins</td><td>&nbsp;</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>&nbsp;</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>&nbsp;</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>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>13-08-2012</td><td>Ligations</td><td>Victor</td><td>"nFF GFP XP GE + 1A3 XP GE</td></tr> <tr><td>nFF GFP XP GE + 1A3 XP GE</td></tr> <tr><td>nFF GFP XP GE + T7 GE SP</td></tr> <tr><td>nFF GFP XP GE + K314100 GE SP</td></tr> <tr><td>nFF GFP ctrl"</td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>13-08-2012</td><td>Digestions </td><td>Victor</td><td>nFF GFP GE (3) with XP</td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>13-08-2012</td><td>Ligations</td><td>Kevin</td><td>"Vectors: K314104, K081005, K314100, K314003, J04500</td></tr> <tr><td>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</td></tr> <tr><td>nFF GFP GE with XP"</td><td>"digested MPP for 13 mins at 37C</td></tr> <tr><td>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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>14-08-2012</td><td>Digestions </td><td>David </td><td>GEs SmtA + FliC, fMt + FliC1 with SpeI</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>14-08-2012</td><td>Digestions </td><td>Victor</td><td>nFF GFP GE with XP</td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</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>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</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>&nbsp;</td><td>&nbsp;</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 </td></tr> <tr><td>AK 1:1 100 uL - 10</td></tr> <tr><td>AK 1:1 the rest - 21</td></tr> <tr><td>K 1:1 100 uL - 50</td></tr> <tr><td>AK: 1:3 100 uL - 0</td></tr> <tr><td>AK 1:3 the rest - 2</td></tr> <tr><td>K 1:3 100 uL - 2"</td><td>&nbsp;</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>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>15-08-2012</td><td>Gel</td><td>Victor</td><td>"DIG B0015 EX</td></tr> <tr><td>DIG B0017 EX</td></tr> <tr><td>DIG nFF GFP + J04500 ES</td></tr> <tr><td>pFF GFP + 1A3</td></tr> <tr><td>MPP nFF GFP + J04506</td></tr> <tr><td>MPP nFF GFP + J04560"</td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</td></tr> <tr><td>15-08-2012</td><td>Gel</td><td>Phillip</td><td>"MPP nFF + J04500</td></tr> <tr><td>MPP pFF GFP 1A3"</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>15-08-2012</td><td>Liquid culture</td><td>Phillip</td><td>nFF GFP + 1A3 </td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>15-08-2012</td><td>Digestioin</td><td>Victor</td><td>B0015, B0017 terminators with EX</td><td>&nbsp;</td><td>&nbsp;</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</td></tr> <tr><td>extension time 5 min 30 s, 17 cycles"</td><td>&nbsp;</td></tr> <tr><td>15-08-2012</td><td>Liquid Culture</td><td>Kevin</td><td>nFFGFP 1A3 4 trials, J04500</td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</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>&nbsp;</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>&nbsp;</td></tr> <tr><td>16-08-2012</td><td>Digestion</td><td>Victor</td><td>Luciferase PCR products with XS </td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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</td></tr> <tr><td>gelled to check concs: GE nFF GFP XP, GE xylE + RBS XP plasmid, GE xylE + RBS XP"</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>16-08-2012</td><td>PCR overlap extension</td><td>Kevin</td><td>"inserts: mCer, luciferase</td></tr> <tr><td>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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>16-08-2012</td><td>Spectrometer </td><td>Kevin</td><td>&nbsp;</td><td>"OD600</td></tr> <tr><td>2XTY against no reference: 0.055</td></tr> <tr><td>J04500 culture: 1.878</td></tr> <tr><td>nFF GFP + J04500 1:2 K: 1.701 (using a different cuvette = 1.731)</td></tr> <tr><td>nFF GFP + J04500 1:2 AK: 1.760"</td><td>&nbsp;</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>&nbsp;</td></tr> <tr><td>16-08-2012</td><td>Gel</td><td>Phillip</td><td>PCR-OE products: luciferase Kapa, luciferase Phusion</td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</td><td>"use JA-10 rotor and 250 mL bottles</td></tr> <tr><td>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>&nbsp;</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</td></tr> <tr><td>autoclave at Liquid 1 setting"</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>"resuspended cell pellet in 100 mL Tris-HCl</td></tr> <tr><td>blended 1 min 30 s in Waring blender at 4C</td></tr> <tr><td>centrifuged 10 mins. at 4C, 10 000 rpm, speed 6000 </td></tr> <tr><td>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>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>18-08-2012</td><td>Miniprep</td><td>Mitangi</td><td>nFF GFP 1A2, nFF GFP 1C3</td><td>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</td><td>&nbsp;</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>&nbsp;</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>&nbsp;</td><td></td></tr></table>
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  <div id="notebookcontent">
  <div id="notebookcontent">
<p style="font-size: 2em; text-align:middle'"> Given that the vast majority of our work now is in the lab, we will only be updating the "Labwork" section of the Notebook. </p>
<p style="font-size: 2em; text-align:middle'"> Given that the vast majority of our work now is in the lab, we will only be updating the "Labwork" section of the Notebook. </p>

Revision as of 03:55, 4 October 2012
Control

Notebook - Week 1

DateProtocolPeopleDNA (if relevant)Quantities and Parameters (if relevant)Notes on Protocol
12-08-2012PCRBeiniGE pFliC1 + GFP + FliC2  
12-08-2012Digestion with XSFaisal PCR products SmtA and fMt Fast Digest, enzymatic purification using Biobasic kit
12-08-2012PCR overlap extension of MBPs with FliC1FaisalSmtA, fMt, FliC1  
12-08-2012Soft agar (0.25%) plates Beini made 7 plates each of [C], [amp], [tet] 
12-08-2012MiniprepBeiniK314100 high exp cassette, K314101 low exp cassette, K314103 lac expression cassette  
12-08-2012GelBeiniK081005 SP GE, K314104 SP GE  
12-08-2012GelBeininFF GFP GE, nFF GFP XP GE, pFF GFP XP GE, MPP K314100, MPP K314101, MPP K314103  
12-08-2012Gel extractionKevinnFF GFP, nFF GFP XP, pFF GFP XP, K081005 SP, K314104 SP  
12-08-2012GelKevinMPP K081005, MPP K13104, pFF GFP, nFF GFP, pFF GFP XS  
12-08-2012Digestions Beini"MPP K314100 with SP
MPP K314103 with SP
GE nFF GFP with XP"digested MPPs for 12 mins, GEs for 30 mins 
12-08-2012LigationsKevinpFF GFP in 1A2 from K088006used 6 and 8 uL of vector for 1:1 and 3:1 ligations 
12-08-2012Gel Kevin1:1 lig, 3:1 lig, DIG XP nFF GFP, DIG SP K314100, DIG SP K314103 gel extracted the digestions
12-08-2012Heat Shock Transformation using Motility PlatesKevin   
13-08-2012LigationsVictor"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"  
13-08-2012GelDavid, KevinfMT FliC, SmA FliC, DIG nFF GFP XP, LIG nFF GFP XP 1A3, LIG nFF GFP XP T7, LIG nFF GFP XP K314100  
13-08-2012Digestions VictornFF GFP GE (3) with XP  
13-08-2012Gel extractionDavidfMt FliC, SmtA FliC from above gel  
13-08-2012GelKevinnFF GFP XP, K314100 SP, SmtA FliC1, fMt FliC1  
13-08-2012LigationsKevin"Vectors: K314104, K081005, K314100, K314003, J04500
Insert: nFF GFP XP"1:1, 2:1 insert:vector ratiosplated 100 uL on [K], 100 uL on [AK], spun down the rest at 12000 rpm and resuspended 100 uL on [AK]
13-08-2012Digestions 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
14-08-2012Liquid cultures KevinpFF GFP 1A3 1:3 lig (the rest)   
14-08-2012Digestions David GEs SmtA + FliC, fMt + FliC1 with SpeI  
14-08-2012Digestions VictornFF GFP GE with XP  
14-08-2012Digestions of linearized plasmid backboneVictorpSB1C3, pSB1A3 with XP and DpnI digestion purification
14-08-2012Gel ExtractionVictorDIG XP nFF GFP, npp pFF GFP 1A3, DIG EP pFF GFP 1A3, pSB1C3 XP  did enzymatic purification of pSB1C3 digest instead of gel purification
14-08-2012Gel ExtractionKevinnFF GFP XP, nFF GFP XP, DIG xylE + RBS XP  
14-08-2012MiniprepKevinLIG pFF GFP + 1A3 1:3 (the rest)  
14-08-2012DigestionKevinLIG pFF GFP + 1A3 1:3 (the rest) with EP  
14-08-2012PCR overlap extensionFaisalfliC1 + MBP, fliC2did 1:1, 2:1, 3:1 ratios of fliC1 + MBP : fliC2digestion purification of SmtA + fliC1 and fMT + fliC1 digested with SpeI
14-08-2012LigationsVictor, MitangipSB1A3 + nFF GFPdid 1:1, 2:1 ratios of insert : vector 
14-08-2012Gel to check for concentrationsVictornFF GFP GE XP, nFF GFP GE XP, GE xylE + RBS XP plasmid, GE xylE + RBS  
14-08-2012Liquid cultures of fluorescent coloniesKevin, FaisalnFF 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" 
14-08-2012Heat shock transformationBeininFF GFP + 1A3 1:1, 2:1, ctrl 
14-08-2012Gel extractionFaisal, Davidfmt FliC1 + FliC2, SmtA FliC1 + FliC2  
15-08-2012MiniprepsKevinnFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest  
15-08-2012PCR of Full FliC MBP Gel ExtractionsDavid, Faisalfmt FliC1 + FliC2, SmtA FliC1 + FliC2  
15-08-2012DigestionPhillipnFF GFP + J04500 6 trials, pFF GFP 1A3 1:1 the rest with ES  
15-08-2012GelVictor"DIG B0015 EX
DIG B0017 EX
DIG nFF GFP + J04500 ES
pFF GFP + 1A3
MPP nFF GFP + J04506
MPP nFF GFP + J04560"  
15-08-2012LigationsVictorGE nFF GFP XP, 1C3 XP GEdid 1:1, 2:1 insert:vector ratios  
15-08-2012GelPhillip"MPP nFF + J04500
MPP pFF GFP 1A3"  
15-08-2012Liquid culturePhillipnFF GFP + 1A3   
15-08-2012Streak platingVictornFF GFP + J04500 1:2, 1:3, 1:1 
15-08-2012Gel Extraction of FF MBP products from PCR overlap extensionFaisalSmtA FliC1 + FliC2  
15-08-2012DigestioinVictorB0015, B0017 terminators with EX  
15-08-2012PCR overlap extensionKevinmCer DP XS, nFF GFP + J04500"need a super high insert:vector ratio, >>50:1
extension time 5 min 30 s, 17 cycles" 
15-08-2012Liquid CultureKevinnFFGFP 1A3 4 trials, J04500  
15-08-2012Motility plate inoculation KevinnFF GFP + J04500 6 trials3 uL stab + inject per spot1 stop only on pipette
15-08-2012Digestion of mCer OE KevinmCer OE17 uL DNA, 1 uL DpnI, 2 uL buffer Tango 
16-08-2012Digestion of PP mCer and PP mCit with XSDavidPP mCer, PP mCit ran out of SpeI, only got a bit of S for mCit1; mCer2 and mCit2 with X only
16-08-2012LigationsVictorGE nFF GFP XP + 1C3 XP GE from Antigen-431:1 insert:vector 
16-08-2012DigestionVictorLuciferase PCR products with XS   
16-08-2012MiniprepKevinnFF GFP + 1A3, J04500, nFF GFP + 1A3, nFF GFP + 1A3  
16-08-2012Digestion with EPKevinnFF GFP + 1A3, nFF GFP + 1A3, nFF GFP + 1A3digested 10 uL of dnaNEB
16-08-2012Gel extractionVictor, 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
16-08-2012GelKevinMPP 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  
16-08-2012Digestion w/ EcoRI-HFKevinMPP nFF GFP 1A3 2:1 the rest  
16-08-2012GelKevinGE mCer X, GE luciferase XS, DIG nFF GFP E  
16-08-2012PCR overlap extensionKevin"inserts: mCer, luciferase
vector: nFF GFP + J04500 K"8 mins. 30 s extension time, 18 cycles1 trial with Kapa, 1 trial with Phusion
16-08-2012Liquid culturesKevinfluorescent colony AK nFF GFP J04500 1:3 the rest from 08/14 streak plate  
16-08-2012Spectrometer 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" 
16-08-2012Motility plate inoculationsKevinJ04500, nFF GFP + J04500 1:2 K, nFF GFP + J04500 1:2 AK only J04500 shows motility 
16-08-2012GelPhillipPCR-OE products: luciferase Kapa, luciferase Phusion  
17-08-2012250 mL inoculations of 2XTYVictor AK nFF GFP + J04500 1:2, AK J04500   
17-08-2012Digestions with DpnI using Tango bufferPhillipPCR-OE products: luciferase Kapa, luciferase Phusion  
17-08-2012Using the ultracentrifuge to obtain a bacterial cell pelletVictor, as told by Jun   "use JA-10 rotor and 250 mL bottles
10 000 rpm, 6000 speed, 10 mins., 4C"
17-08-2012Tris-HCl bufferVictor "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" 
17-08-2012Liquid culture of ligationsBeininFF GFP 1C3 100 pellet, nFF GFP 1C3 100 uL, nFF GFP 1A2 1:1 pellet, nFF GFP 1A2 1:1 100 uL  
18-08-2012Isolation of flagellaBeiniAK 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"
18-08-2012Liquid culturesMitangiAK DIG luciferase Kapa DpnI  
18-08-2012MiniprepMitanginFF GFP 1A2, nFF GFP 1C3  
18-08-2012Digestion with PstIMitanginFF GFP 1A2, nFF GFP 1C3, nFF GFP 1A3 (all MPPs)  
18-08-2012GelBeiniMPP 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  
18-08-2012MiniprepBeiniAK nFF GFP J04500 1:3 the rest made glycerol stock
18-08-2012MiniprepBeiniAK 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.

Retrieved from "http://2012.igem.org/Team:Queens_Canada/Notebook/Week16"