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Team:GeorgiaTech/WeekTwelve
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| - | + | <title>10/17-10/23</title><style type="text/css">ol{margin:0;padding:0}p{margin:0}.c13{vertical-align:top;width:114.0pt;border-style:solid;border-color:#000000;border-width:1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt}.c6{vertical-align:top;width:113.25pt;border-style:solid;border-color:#000000;border-width:1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt}.c15{vertical-align:top;width:234.0pt;border-style:solid;border-color:#000000;border-width:1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt}.c0{vertical-align:top;width:117.0pt;border-style:solid;border-color:#000000;border-width:1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt}.c9{vertical-align:sub;color:#ffffff;font-size:6.666666666666666pt;font-family:Arial}.c7{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:72.0pt}.c11{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c17{line-height:1.15;text-indent:36.0pt;direction:ltr}.c3{line-height:1.15;text-indent:0pt;direction:ltr}.c1{color:#ffffff;font-size:10pt;font-family:Arial}.c12{line-height:1.0;text-indent:0pt;direction:ltr}.c2{text-decoration:underline}.c16{border-collapse:collapse}.c4{font-style:italic}.c5{font-weight:bold}.c14{list-style-type:disc}.c10{background-color:#ffffff}.c8{list-style-type:circle}</style></head><body class="c10"><p class="c3"><span class="c1 c5">10/17/2010</span></p><p class="c3"><span class="c1 c2">Goals</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">transformations of NB of ligations done 10/16/2010</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">hyb.ompa.aox</span></li><li class="c7"><span class="c1">hyb.ompa.aoxb</span></li><li class="c7"><span class="c1">hyb.ompa.rfp-f3r</span></li><li class="c7"><span class="c1">negative control</span></li></ol><ol class=""><li class="c11" value="2"><span class="c1">miniprep of aoxb colony b5 </span></li></ol><p class="c17"><span class="c1 c4 c5">See Protocols page for Plasmid DNA Purification with Mini Prep Kit</span></p><ol class=""><li class="c11" value="3"><span class="c1">nanospec the minipreps done 10/16/2010 and today</span></li><li class="c11"><span class="c1">check results of digests on gel</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">digests of hyb.ompa.aoxa/b colonies b1-3, a10 with EcoRI and Spe I</span></li></ol><ol class=""><li class="c11" value="5"><span class="c1">grow up a colony of psb1c3</span></li></ol><p class="c3"><span class="c1 c2 c5"> </span></p><p class="c3"><span class="c1 c2">Protocols</span></p><p class="c3"><span class="c1 c2"> </span></p><p class="c3"><span class="c1 c2">transformations of NB using ligations done 10/16/2010 (Christina)</span></p><ol class="c8"><li class="c7" value="1"><span class="c1">hyb.ompa.aox</span></li><li class="c7"><span class="c1">hyb.ompa.aoxb</span></li><li class="c7"><span class="c1">hyb.ompa.rfp-f3r</span></li><li class="c7"><span class="c1">negative control</span></li></ol><p class="c3"><span class="c1">10 ul of NB + 5 ul of ligation rxn</span></p><p class="c3"><span class="c1">plate at 1 pm </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">gel of digests hyb.ompa.aoxa/b colonies b1-3, a10 with EcoRI and Spe I (Scott)</span></p><p class="c3"><span class="c1">Note: problem with wells 2,3</span></p><p class="c3"><span class="c1 c4">order: ladder|b1|b2|b3|a10|b1|b2|ladder (exacto)</span></p><p class="c3"><span class="c1">Colony aoxa A10 has the pattern we want: a 1 kb insert adn 2 kb bakbone</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">miniprep of colony b5 (Scott)</span></p><p class="c3"><span class="c1 c4 c5">See Protocols page for Plasmid DNA Purification with Mini Prep Kit</span></p><p class="c3"><span class="c1 c2">nanospec</span></p><p class="c3"><span class="c1">hyb.ompa.aox.psb1a3 colony b5 (10.16.2010)=90 ng/uL</span></p><p class="c3"><span class="c1">hyb.ompa.aox.psb1a3 colony b5 (10.17.2010)=101 ng/uL</span></p><p class="c3"><span class="c1 c2"> </span></p><p class="c3"><span class="c1 c2">Repeat pcrs to make hyb-Fr, RFP-FR, aox1b FR, aox1a FR</span></p><p class="c3"><span class="c1 c5">Rxns </span></p><p class="c3"><span class="c1 c5">Hyb-F and Hyb-R (note use 1 ul of template for hyb)</span></p><p class="c3"><span class="c1 c5">RFP-F and RFP-R</span></p><p class="c3"><span class="c1 c5">Aox1b-F and Aox1b-R</span></p><p class="c3"><span class="c1 c5">aox1a-F and aox1a-R</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">PCR Protocol </span><span class="c1"> </span></p><p class="c3"><span class="c1">26.5 uL H2O -</span></p><p class="c3"><span class="c1">10 uL </span><span class="c1 c5">PHUSION 5X Reaction buffer -</span></p><p class="c3"><span class="c1">5 uL forward primer -</span></p><p class="c3"><span class="c1">5 uL reverse primer -</span></p><p class="c3"><span class="c1">1 uL dNTP 10 mM - (thawed & kept on ice)</span></p><p class="c3"><span class="c1 c5">2 uL</span><span class="c1"> template DNA (note: use 1 uL for hybB) - </span></p><p class="c3"><span class="c1">0.5 uL polymerase enzyme, </span><span class="c1 c5">PHUSION -</span></p><p class="c3"><span class="c1 c5">Total Volume= 50 uL</span></p><p class="c3"><span class="c1 c2"> </span></p><p class="c3"><span class="c1 c2">grow up a starter colony of psb1c3 (Christina)</span></p><p class="c3"><span class="c1">The colonies were pinkish, so Christina picked a colony so we can miniprep them tomorrow</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">Digest of colony B5 of hyb.ompa.aoxb.psb1a3 (from 10.16.2010)(Margo)</span></p><p class="c3"><span class="c1 c5">colony 5, Aox1b colony PCR from 10-16: Label on tube in freezer is 5</span><span class="c9 c5">16</span></p><p class="c3"><span class="c1">4.5 uL H20 </span></p><p class="c3"><span class="c1">2uL 10X EcoRI buffer</span></p><p class="c3"><span class="c1">10 uL hyb.ompa.aoxb.psb1a3 colony 5 miniprep (10.16.2010)</span></p><p class="c3"><span class="c1">2 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c3"><span class="c1">0.75 uL SpeI</span></p><p class="c3"><span class="c1">0.75 uL EcoRI</span></p><p class="c3"><span class="c1 c5">Total=20 ul total </span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c5">colony 5, Aox1b colony PCR from 10-17: Label on tube in freezer is 5</span><span class="c5 c9">17</span></p><p class="c3"><span class="c1">4.5 uL H20 </span></p><p class="c3"><span class="c1">2uL 10X EcoRI buffer</span></p><p class="c3"><span class="c1">10 uL hyb.ompa.aoxb.psb1a3 colony 5 miniprep (10.17.2010)</span></p><p class="c3"><span class="c1">2 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c3"><span class="c1">0.75 uL SpeI</span></p><p class="c3"><span class="c1">0.75 uL EcoRI</span></p><p class="c3"><span class="c1 c5">Total=20 ul total </span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c5">Into heat block at 1:51 p.m., remove at 4:51 p.m.</span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c2">Making Gels</span></p><p class="c3"><span class="c1">Started at 2:26 pm. Gels ready at 3:26 pm. </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Gels:</span></p><ol class=""><li class="c11" value="1"><span class="c1">4 ul of the PCRs Christina did today </span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">aoxa FR</span></li><li class="c7"><span class="c1">aoxb FR</span></li><li class="c7"><span class="c1">hyb FR</span></li><li class="c7"><span class="c1">RFp FR</span></li></ol><ol class="c14"><li class="c11" value="2"><span class="c1">RE digest 5</span><span class="c9">16</span></li><li class="c11"><span class="c1">RE digest 5</span><span class="c9">17</span></li></ol><p class="c3"><span class="c1">Gel started 5:00 pm. </span></p><p class="c3"><span class="c1">Results: </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c5">10/18/2010</span></p><p class="c3"><span class="c1 c2">Goals</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">Send off for sequencing the minirep of colony a10 </span></li></ol><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">Colony PCR of hyb.ompa+aoxa+psb1a3, hyb.ompa+aoxb+psb1a3, hyb.ompa+RFP-F3R+psb1a3</span></p><p class="c3"><span class="c1">2.5 uL of cells</span></p><p class="c3"><span class="c1">11.75 uL H2O</span></p><p class="c3"><span class="c1">5 uL GOTAQ</span><span class="c1 c5"> 5X Reaction buffer</span></p><p class="c3"><span class="c1">2.5 uL Hyb-F forward primer</span></p><p class="c3"><span class="c1">2.5 uL Ompa-R reverse primer</span></p><p class="c3"><span class="c1">.5 uL dNTP 10 mM - (thawed & kept on ice)</span></p><p class="c3"><span class="c1">0.25 uL polymerase enzyme, TAQ</span></p><p class="c3"><span class="c1 c5">Total Volume= 25 uL </span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1">Picked four white colonies from each plate, added to 25uL water. </span></p><p class="c3"><span class="c1"> </span></p><table cellpadding="0" cellspacing="0" class="c16"><tbody><tr><td class="c15"><p class="c12"><span class="c1 c5">Plate</span></p></td><td class="c15"><p class="c12"><span class="c1 c5">Microcentrifuge Tube Numbers</span></p></td></tr><tr><td class="c15"><p class="c12"><span class="c1">psB1A3-aox1A (negative control, 10/17)</span></p></td><td class="c15"><p class="c12"><span class="c1">1,2,3,4</span></p></td></tr><tr><td class="c15"><p class="c12"><span class="c1">psB1A3-aox1B-hybB-ompA (10/17)</span></p></td><td class="c15"><p class="c12"><span class="c1">5,6,7,8</span></p></td></tr><tr><td class="c15"><p class="c12"><span class="c1">psB1A3-aox1A-hybB-ompA (10/17)</span></p></td><td class="c15"><p class="c12"><span class="c1">9,10,11,12</span></p></td></tr><tr><td class="c15"><p class="c12"><span class="c1">psB1A3-hybB-ompA-RFP,F3R (10/17)</span></p></td><td class="c15"><p class="c12"><span class="c1">13,14,15,16</span></p></td></tr></tbody></table><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">Run gel on colony PCRS from above. </span></p><p class="c3"><span class="c1">Lanes were run in order (1-16), with a 1KB ladder middle band (there was only 2uL of this left, so the ladder is faint).</span></p><p class="c3"><img height="330.0" src="https://static.igem.org/mediawiki/2010/4/47/10-17.0.png" width="440.0"></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">Gel electrophoresis of Top and Bottom bands from 17 OCT 2010</span></p><p class="c3"><img height="0.0" src="images/" width="0.0"></p><p class="c3"><img height="324.0" src="https://static.igem.org/mediawiki/2010/c/c4/10-17.12.png" width="433.0"></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Ladder | Top | Bottom (yes, both are very faint)</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><img height="329.0" src="https://static.igem.org/mediawiki/2010/7/7a/10-17.10.png" width="440.0"></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Ladder | Top | Bottom</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">Retransformation of Ligations from 10-16</span></p><p class="c3"><span class="c1">Repeated heat shock transformations of ligations from 10-16-10. </span></p><p class="c3"><span class="c1">Used 5uL of ligation reaction + 10uL of NB cells. </span></p><p class="c3"><span class="c1">Plated 100uL of each on each plate.</span></p><p class="c3"><span class="c1 c4 c5">See Protocols page for Heat Shock Transformation</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c5">10/19/2010</span></p><p class="c3"><span class="c1 c2">Goals</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">reload colony pcrs from 10/18/2010 if need be (the ladder is faint, so if no interpretation is possible, then load again. otherwise no need to) </span></li><li class="c11"><span class="c1">Load digest the 2 digests of colony B5 hyb.ompa.aoxb.psb1a3 from 10.17.2010 (done)</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">if they have the expected insert, send off for sequencing</span></li></ol><ol class=""><li class="c11" value="3"><span class="c1">Load the pcrs from 10.17 of hyb-FR, aoxa-Fr, aoxb-FR, and rfp-F3R on a gel (done)</span></li><li class="c11"><span class="c1">pick (20) colonies of RFP-F3 from 10/18/2010 (done)</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">after the colony pcr, add 3ml of LB instead of 250 ul. This will prepare us for minipreps tomorrow.</span></li></ol><ol class=""><li class="c11" value="5"><span class="c1">Pick (20) colonies of aoxb (from 10.18/2010 plates) (done)</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">after the colony pcr, add 3ml of LB instead of 250 ul. This will prepare us for minipreps tomorrow.</span></li></ol><ol class=""><li class="c11" value="6"><span class="c1">try the ligation of hyb-rfp-psb1a3 , transform into NB or bl21</span></li><li class="c11"><span class="c1">transform the “sequenced” colonies (a10) into bl21</span></li><li class="c11"><span class="c1">miniprep of psb1C3 (done)</span></li><li class="c11"><span class="c1">Send colony a10 for sequencing (Christina)(to be done)</span></li><li class="c11"><span class="c1">grow up some more colony a10 from glycerol stock</span></li></ol><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">Protocols</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">loading gel (done, Scott)</span></p><p class="c3"><span class="c1 c4">order:</span></p><p class="c3"><span class="c1 c4">exact ladder|aoxa-FR phusion pcr (10.17.2010)||aoxb-FR phusion pcr (10.17.2010)||hyb-FR phusion pcr (10.17.2010)||RFP-F3R phusion pcr (10.17.2010)|exact ladder| digest of miniprep of colony b5 (10.16 version) hyb.omp.aoxb.psb1a3 from 10.17.2010|digest of miniprep of colony b5 (10.17 version) hyb.omp.aoxb.psb1a3 from 10.17.2010</span></p><p class="c3"><span class="c1 c4">start 11:08 am</span></p><p class="c3"><span class="c1">the pcrs appeared to work, but the digests do not contain the predicted band pattern.</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c2">Miniprep of psb1C3(Scott) (done)</span></p><p class="c3"><span class="c1 c4 c5">See Protocols page for Plasmid DNA Purification with Mini Prep Kit</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c2">Prepare DNA to send off for sequencing.( Christina)(Scott)</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">In standard PCR tubes, prepare template DNA with just water and ONE primer (forward or reverse). The total volume should be 15 uL. The concentration of DNA in the sample should be adjusted to match the length of the template DNA; there is a chart in the Gaucher lab on the wall above the EtBr disposal bucket. For example, for a PCR product 1000 to 2000 bp in length, the concentration of the sample should be ~ 4 ng/uL. </span></p><p class="c3"><span class="c1 c5">Predicted size of hybB-ompA-aox1a: 1509 bp. Calculate concentration to be ~ 4 ng/uL</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">An email should be sent to Ryan with the following information:</span></p><p class="c3"><span class="c1">Tube Label: actually written on the tube, in this form: RR01, RR02, etc.</span></p><p class="c3"><span class="c1">DNA Name: this is something meaningful to us, like AOXa_construct_PSB1A3_vector_1</span></p><p class="c3"><span class="c1">DNA Type: Purified PCR or Plasmid</span></p><p class="c3"><span class="c1">DNA length: in bp</span></p><p class="c3"><span class="c1">My primer: a name meaningful to us</span></p><p class="c3"><span class="c1">Give the tubes to Ryan. Apparently they should not be frozen, just kept on ice.</span></p><p class="c3"><span class="c1 c4">Rxns: colony 10 hyb.ompa.aoxa miniprep from 10/15/2010</span></p><p class="c3"><span class="c1 c4">psb1a3-F</span></p><p class="c3"><span class="c1 c4">hybF</span></p><p class="c3"><span class="c1 c4">aox-F</span></p><p class="c3"><span class="c1 c4">aox-middle</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c2">colony PCRs (Rob, Christian, Scott)</span></p><p class="c3"><span class="c1">use plates from 10/18/2010</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c4">use hyb-f and ompa-r</span></p><p class="c3"><span class="c1 c4">Reactions:</span></p><p class="c3"><span class="c1">aoxa 1-20</span></p><p class="c3"><span class="c1">aoxb 1-20</span></p><p class="c3"><span class="c1">rfp-f3r 1-20</span></p><p class="c3"><span class="c1 c2"> </span></p><p class="c3"><span class="c1 c2">Colony pcr Master mix (batch of 70 amount in brackets)</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">11.75 uL H2O (822.5 uL)-</span></p><p class="c3"><span class="c1">5 uL GOTAQ</span><span class="c1 c5"> 5X Reaction buffer </span><span class="c1">(350 uL)-</span></p><p class="c3"><span class="c1">2.5 uL Hyb-F forward primer (175 uL)-</span></p><p class="c3"><span class="c1">2.5 uL Ompa-R reverse primer (175 uL)-</span></p><p class="c3"><span class="c1">.5 uL dNTP 10 mM - (thawed & kept on ice) (35 uL)-</span></p><p class="c3"><span class="c1">0.25 uL polymerase enzyme, TAQ (17.5 uL)</span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1">2.5 uL of cells added to each respective PCR tube</span></p><p class="c3"><span class="c1 c5">Total Volume= 25 uL </span></p><p class="c3"><span class="c1 c5">Start: 3:31 pm</span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c5">10/20/2010</span></p><p class="c3"><span class="c1 c2">goals</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">load rest of colony pcrs on gel from 10/19</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">load 5 ul</span></li></ol><p class="c3"><span class="c1 c2">protocols</span></p><p class="c3"><span class="c1 c2"> </span></p><p class="c3"><span class="c1 c2">loading gels for colony pcrs (Scott)</span></p><p class="c3"><span class="c1">expected fragments:</span></p><p class="c3"><span class="c1">hyb-f and ompa-r approx 500 bp. </span></p><p class="c3"><span class="c1">gel one (started 9:36 am)</span></p><p class="c3"><span class="c1 c4">order:</span></p><p class="c3"><span class="c1 c4">exact ladder|aoxa colony pcrs 1-6|ladder|aoxa colony pcrs 7-14|ladder</span></p><p class="c3"><img height="330.0" src="https://static.igem.org/mediawiki/2010/1/18/10-17.3.png" width="441.0"></p><p class="c3"><span class="c1">gel two</span></p><p class="c3"><span class="c1 c4">exact ladder|aoxa colony pcrs 15-20|colony pcr aoxb 1|ladder|two empty wells|ladder|aoxb colony pcrs 2-6|</span></p><p class="c3"><img height="334.0" src="https://static.igem.org/mediawiki/2010/d/de/10-17.4.png" width="447.0"></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c4">gel three</span></p><p class="c3"><span class="c1 c4">order</span></p><p class="c3"><span class="c1 c4">exact ladder|aoxb colony pcrs 7-12 (from 10.19.2010)|ladder</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c4">gel four</span></p><p class="c3"><span class="c1 c4">order</span></p><p class="c3"><span class="c1 c4">exact ladder|aoxb colony pcrs 13-18 (from 10.19.2010)|ladder</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c4">(gel 3 and 4 are together below)</span></p><p class="c3"><img height="330.0" src="https://static.igem.org/mediawiki/2010/0/0e/10-17.11.png" width="441.0"></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c4">gel five</span></p><p class="c3"><span class="c1 c4">order</span></p><p class="c3"><span class="c1 c4">exact ladder|aoxb colony pcrs 19-20 (from 10.19.2010)|rfp-f3r colonies 1-4ladder</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c4">gel six</span></p><p class="c3"><span class="c1 c4">order</span></p><p class="c3"><span class="c1 c4">exact ladder|rfp-f3r colony pcrs 5-10(from 10.19.2010)|ladder</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c4">(gel 5 and 6 are below)</span></p><p class="c3"><img height="329.0" src="https://static.igem.org/mediawiki/2010/9/95/10-17.8.png" width="441.0"></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c4">gel seven</span></p><p class="c3"><span class="c1 c4">order</span></p><p class="c3"><span class="c1 c4">exact ladder|rfp-f3r colony pcrs 10-20(from 10.19.2010)|ladder</span></p><p class="c3"><img height="329.0" src="https://static.igem.org/mediawiki/2010/3/32/10-17.2.png" width="440.0"></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">RE Double Digest Recipe for pSB1C3 miniprep (from 10/19/2010) -- digest with speI and EcorI (Debika)</span></p><p class="c3"><span class="c1 c2">STARTED AT 2.54 PM</span></p><p class="c3"><span class="c1">12.5 uL H20 - </span></p><p class="c3"><span class="c1">3uL EcoRI Buffer -</span></p><p class="c3"><span class="c1">10 uL pSB1C3 (10.8.2010, 56 ng/uL)</span></p><p class="c3"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml) -</span></p><p class="c3"><span class="c1">0.75 uL SpeI</span></p><p class="c3"><span class="c1">0.75 uL EcoRI</span></p><p class="c3"><span class="c1 c5">Total=30 ul total</span></p><p class="c3"><span class="c1">Digest overnight - put in waterbath 37 degrees at 2.54 PM</span></p><p class="c3"><span class="c1">Predict a insert (1000 bp) an(?- this was already there from the last protocol that Scott copied for me, DM)</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c5">Things to do</span></p><p class="c3"><span class="c1 c5">1. load colonies rfp-f3r 10-20 on gel, visualize for 500 bp band (done Christina)</span></p><p class="c3"><span class="c1 c5">2. colony pcr using aoxa/b f-R from colonies on 10/19/2010 (Done Christina)</span></p><p class="c3"><span class="c1 c5">3. grow up good colonies in 5ml liq culture</span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c2">colony pcr using aoxa/b f-R from colonies on 10/19/2010 </span></p><p class="c3"><span class="c1 c4">Do 2nd pcr on colonies (from 10/19/2010)</span></p><p class="c3"><span class="c1 c4">aoxa: 3,5,6</span></p><p class="c3"><span class="c1 c4">aoxb: 15,18</span></p><p class="c3"><span class="c1 c4">rfp-f3: 1, 11, 12, 18, 19</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Note: change the colony pcr program to have an extension time of 1.5 mins. </span></p><p class="c3"><span class="c1 c5">For aoxa (colonies 3, 5,6)</span></p><p class="c3"><span class="c1">11.75 uL H2O </span></p><p class="c3"><span class="c1">5 uL GOTAQ</span><span class="c1 c5"> 5X Reaction buffer </span></p><p class="c3"><span class="c1">2.5 uL Hyb F forward primer </span></p><p class="c3"><span class="c1">2.5 uL Aoxa R reverse primer </span></p><p class="c3"><span class="c1">.5 uL dNTP 10 mM - (thawed & kept on ice) </span></p><p class="c3"><span class="c1">0.25 uL polymerase enzyme, TAQ </span></p><p class="c3"><span class="c1">.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube</span></p><p class="c3"><span class="c1 c5">Total Volume= 25 uL </span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c5">For aoxb (colonies 15, 18)</span></p><p class="c3"><span class="c1">11.75 uL H2O </span></p><p class="c3"><span class="c1">5 uL GOTAQ</span><span class="c1 c5"> 5X Reaction buffer </span></p><p class="c3"><span class="c1">2.5 uL Hyb F forward primer </span></p><p class="c3"><span class="c1">2.5 uL Aoxb R reverse primer </span></p><p class="c3"><span class="c1">.5 uL dNTP 10 mM - (thawed & kept on ice) </span></p><p class="c3"><span class="c1">0.25 uL polymerase enzyme, TAQ </span></p><p class="c3"><span class="c1">.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube</span></p><p class="c3"><span class="c1 c5">Total Volume= 25 uL </span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c5">For RFP f3r (colonies 1, 11, 12, 18, 19)</span></p><p class="c3"><span class="c1">11.75 uL H2O </span></p><p class="c3"><span class="c1">5 uL GOTAQ</span><span class="c1 c5"> 5X Reaction buffer </span></p><p class="c3"><span class="c1">2.5 uL HybF forward primer </span></p><p class="c3"><span class="c1">2.5 uL Rfp-r reverse primer </span></p><p class="c3"><span class="c1">.5 uL dNTP 10 mM - (thawed & kept on ice) </span></p><p class="c3"><span class="c1">0.25 uL polymerase enzyme, TAQ </span></p><p class="c3"><span class="c1">.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube</span></p><p class="c3"><span class="c1 c5">Total Volume= 25 uL </span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c5">*NOTE - can use the PCR reaction (.25uL) from today on aoxa 3,5,6; aoxb 15, 18; rfp-f3 1 as template DNA. (good point)</span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c2">Ligation of RFP-FR,HybB-FR,to psb1a3 (Christina)</span></p><p class="c3"><span class="c1 c4">Calculating equivalents: 9C</span></p><p class="c3"><span class="c1 c4">rfp (from9.22.2010)</span></p><p class="c3"><span class="c1 c4">psb1a3 (10.15, top )</span></p><p class="c3"><span class="c1 c4">hyb (from 9.10, purified 10.20)</span></p><p class="c3"><span class="c1">RFP- [41ng/uL]/678 bp= 0.06eq/uL</span></p><p class="c3"><span class="c1">hyBb-[ 6 ng/uL]/393 bp = 0.0152 eq/uL</span></p><p class="c3"><span class="c1">psb1a3-[ 9.4 ng/ul]/2000 bp= 0.0047 eq/uL</span></p><p class="c3"><span class="c1">for linear ligations, use a 2:2:1 of insert:insert:vector </span></p><p class="c3"><span class="c1 c2"> </span></p><p class="c3"><span class="c1">0.5 uL of RFP FR </span></p><p class="c3"><span class="c1">2 uL of HybB FR </span></p><p class="c3"><span class="c1">3 ul psb1a3</span></p><p class="c3"><span class="c1">1 uL 10x Ligase Buffer</span></p><p class="c3"><span class="c1">3 uL H20</span></p><p class="c3"><span class="c1">0.5 uL T4 Ligase</span></p><p class="c3"><span class="c1 c5">Total=10 uL</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">overnight 4c </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">for tomorrow</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">look on list for any overlaps and missed items</span></li><li class="c11"><span class="c1">note: make glycerol stocks when doing minipreps</span></li></ol><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c5">10/21/2010</span></p><p class="c3"><span class="c1 c2">goals</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">glycerol stocks of liquid cultures (done)</span></li><li class="c11"><span class="c1">repeat pcr from 10/20 checking the “good” colonies; the gel showed no predicted bands, but that may be due to pcr errors, elongation time etc. Try using aoxa/b f-r only, or try troubleshooting why the pcr did not make the 1.5 kb band</span></li><li class="c11"><span class="c1">miniprep</span><span class="c1 c5"> </span><span class="c1">liq cultures of confirmed colonies (Scott)(done)</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">nanospec’ed</span></li></ol><ol class=""><li class="c11" value="4"><span class="c1">send ones that are confirmed for sequencing</span></li><li class="c11"><span class="c1">digest the three inserts from 3 good colonies and ligate to psb1c3</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">make sure to pcr purify the digest from yesrerday</span></li></ol><ol class=""><li class="c11" value="6"><span class="c1">transform bl21 using hyb-rfp ligation</span></li><li class="c11"><span class="c1">transform bl21 using minipreps of good colonies</span></li><li class="c11"><span class="c1">check list for anything else!</span></li></ol><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">protocols</span></p><p class="c3"><span class="c1 c2"> </span></p><p class="c3"><span class="c1 c2">glycerol stocks of liq cultures(Christian)</span></p><p class="c3"><span class="c1">colonies:</span></p><p class="c3"><span class="c1 c4">aoxa: 3,5,6</span></p><p class="c3"><span class="c1 c4">aoxb: 15,18</span></p><p class="c3"><span class="c1 c4">rfp-f3: 1, 11, 12, 18, 19</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c2">loading gel to check pcrs yesterday (Scott)</span></p><p class="c3"><span class="c1">rxns:</span></p><p class="c3"><span class="c1">colony pcr using aoxa/b f-R from colonies on 10/19/2010</span></p><p class="c3"><span class="c1 c4">aoxa using hyb-f and aoxa-r: 3,5,6</span></p><p class="c3"><span class="c1 c4">aoxb using hyb-f and aoxb-r: 15,18</span></p><p class="c3"><span class="c1 c4">rfp-f3 using hyb-f and rfp-r: 1, 11, 12, 18, 19</span><span class="c1"> (used hyb-f and rfp-r)</span></p><p class="c3"><img height="334.0" src="https://static.igem.org/mediawiki/2010/1/12/10-17.9.png" width="440.0"></p><p class="c3"><span class="c1 c4">order</span></p><p class="c3"><span class="c1 c4">exact ladder|aoxa colony 3,5,6|aoxb colony 15,18,|rfp-f3r colony1|ladder|rfp-f3r colony 11,12,18,19|ladder</span><br/></p><p class="c3"><span class="c1">version 2 of above gel:</span><br/><img height="309.0" src="https://static.igem.org/mediawiki/2010/5/55/10-17.1.png" width="440.0"></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">RE Double Digest Recipe miniprep (from 10/21/2010) -- digest with speI and EcoRI</span></p><p class="c3"><span class="c1">12.5 uL H20 -</span></p><p class="c3"><span class="c1">3uL EcoRI Buffer -</span></p><p class="c3"><span class="c1">5 uL miniprep -</span></p><p class="c3"><span class="c1">3 uL BSA (1ug/uL, to a final conc of .1mg/ml) -</span></p><p class="c3"><span class="c1">0.75 uL SpeI</span></p><p class="c3"><span class="c1">0.75 uL EcoRI</span></p><p class="c3"><span class="c1 c5">Total=30 ul total</span></p><p class="c3"><span class="c1">3 hour digest</span></p><p class="c3"><span class="c1">started 1pm</span></p><p class="c3"><span class="c1">predicted sizes:</span></p><p class="c3"><span class="c1">2kb (backbone)</span></p><p class="c3"><span class="c1">hyb.ompa./aoxa/b insert approx 1kb</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">pcr using aoxa/b FR (done)</span></p><p class="c3"><span class="c1">rxns</span></p><p class="c3"><span class="c1 c4">aoxa using aoxa-f and aoxa-r: 3,5,6-</span></p><p class="c3"><span class="c1 c4">aoxb using aoxb-f and aoxb-r: 15,18-</span></p><p class="c3"><span class="c1 c4">rfp-f3 using rfp-f and rfp-r: 1, 11, 12, 18, 19-</span><span class="c1"> (used hyb-f and rfp-r)</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">26.5 uL H2O-</span></li><li class="c11"><span class="c1">10 uL </span><span class="c1 c5">Taq 5X Reaction buffer-</span></li><li class="c11"><span class="c1">5 uL forward primer-</span></li><li class="c11"><span class="c1">5 uL reverse primer-</span></li><li class="c11"><span class="c1">1 uL dNTP 10 mM - (thawed & kept on ice)-</span></li><li class="c11"><span class="c1 c5">2 uL</span><span class="c1"> template DNA-</span></li><li class="c11"><span class="c1">0.5 uL polymerase enzyme, </span><span class="c1 c5">Gotaq</span></li></ol><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c5">Total Volume= 50 uL</span></p><p class="c3"><span class="c1">started 1:30pm</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">prepare DNA to send off for sequencing.( Margo)</span></p><p class="c3"><span class="c1">In standard PCR tubes, prepare template DNA with just water and ONE primer (forward or reverse). The total volume should be 15 uL. The concentration of DNA in the sample should be adjusted to match the length of the template DNA; there is a chart in the Gaucher lab on the wall above the EtBr disposal bucket. For example, for a PCR product 1000 to 2000 bp in length, the concentration of the sample should be ~ 4 ng/uL.</span></p><p class="c3"><span class="c1 c5">Predicted size of hybB-ompA-aox1a: 1509 bp. Calculate concentration to be (check sheet)</span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1">An email should be sent to Ryan with the following information:</span></p><p class="c3"><span class="c1">Tube Label: actually written on the tube, in this form: RR01, RR02, etc.</span></p><p class="c3"><span class="c1">DNA Name: this is something meaningful to us, like AOXa_construct_PSB1A3_vector_1</span></p><p class="c3"><span class="c1">DNA Type: Purified PCR or Plasmid</span></p><p class="c3"><span class="c1">DNA length: in bp</span></p><p class="c3"><span class="c1">My primer: a name meaningful to us</span></p><p class="c3"><span class="c1">Give the tubes to Ryan. Apparently they should not be frozen, just kept on ice.</span></p><p class="c3"><span class="c1 c4">Rxns: colony aox5(a5) , aoxb 18(18b), rfp-f3r colony 1 (1r)</span></p><p class="c3"><span class="c1 c4">psb1a3-F</span></p><p class="c3"><span class="c1 c4">hybF</span></p><p class="c3"><span class="c1 c4">aox-F</span></p><p class="c3"><span class="c1 c4">aox-middle</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c5">RR01 = Aox1a colony A5, with psb1a3 forward primer</span></p><p class="c3"><span class="c1 c5">RR02 = Aox1a colony A5, with AOX1a forward primer</span></p><p class="c3"><span class="c1 c5">RR03 = Aox1a colony A5, with AOX1a middle forward primer</span></p><p class="c3"><span class="c1 c5">RR04 = Aox1a colony B18, with psb1a3 forward primer</span></p><p class="c3"><span class="c1 c5">RR05 = Aox1a colony B18, with AOX1b forward primer</span></p><p class="c3"><span class="c1 c5">RR06 = Aox1a colony B18, with AOX1b middle forward primer</span></p><p class="c3"><span class="c1 c5">RR07 = RFP colony R12, with psb1a3 forward primer</span></p><p class="c3"><span class="c1 c5">RR08 = RFP colony R12, with RFP3 forward primer</span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1">RR01 AOX1a_colonyA5_psb1a3_primer 1500 bp psb1A3_forward</span></p><p class="c3"><span class="c1">RR02 AOX1a_colonyA5_aox1af_primer 1500 bp aox1a_forward</span></p><p class="c3"><span class="c1">RR03 AOX1a_colonyA5_aox1amid_primer 1500 bp aox1amid_forward</span></p><p class="c3"><span class="c1">RR04 AOX1b_colonyB18_psb1a3_primer 1500 bp psb1A3_forward</span></p><p class="c3"><span class="c1">RR05 AOX1b_colonyB18_aox1bf_primer 1500 bp aox1b_forward</span></p><p class="c3"><span class="c1">RR06 AOX1b_colonyB18_aox1bmid_primer 1500 bp aox1bmid_forward</span></p><p class="c3"><span class="c1">RR07 RFP_colonyR12_psb1a3_primer 1200 bp psb1A3_forward</span></p><p class="c3"><span class="c1">RR08 RFP_colonyR12_RFP3_primer 1200 bp RFP3_forward</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c4">aoxa using aoxa-f and aoxa-r: 3,5,6-</span></p><p class="c3"><span class="c1 c4">aoxb using aoxb-f and aoxb-r: 15,18-</span></p><p class="c3"><span class="c1 c4">rfp-f3 using rfp-f and rfp-r: 1, 11, 12, 18, 19-</span><span class="c1"> (used hyb-f and rfp-r)</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2 c5">loading gel</span></p><p class="c3"><span class="c1 c2 c5">gel 1 and 2 are of digests using ecoRI and SpeO</span></p><p class="c3"><span class="c1 c5">Gel 1(left)</span></p><p class="c3"><span class="c1 c4">ladder|A3|A5|A6|B15|B18|empty|ladder</span></p><p class="c3"><span class="c1 c5">Gel 2(right)</span></p><p class="c3"><span class="c1 c4">ladder|R1|R11|R12|R18|R19|empty|ladder</span></p><p class="c3"><img height="379.0" src="https://static.igem.org/mediawiki/2010/6/65/10-17.7.png" width="443.0"></p><p class="c3"><span class="c1 c5">gel 3</span></p><p class="c3"><span class="c1 c5">PCR from 10.21.2010 using aoxa/b-FR or rfp-FR (for Rfp-f3r constructs)</span></p><p class="c3"><span class="c1 c5">of colonies from hyb.ompa.aoxa/b or rfp-f3r colonies</span></p><p class="c3"><img height="322.0" src="https://static.igem.org/mediawiki/2010/8/84/10-17.6.png" width="439.0"></p><p class="c3"><span class="c1 c4">order</span></p><p class="c3"><span class="c1 c4">ladder|aox 3a|aox5a|aox 6a|aox 15b|aox 18b|rfp-f3r 1R|ladder| rfp-f3r 11R| rfp-f3r 19R|rfp-f3r18r|ladder</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c2 c5">transformations into bl21: (Christina, Christian)</span></p><p class="c3"><span class="c1 c4">aoxa: 3,5,6-</span></p><p class="c3"><span class="c1 c4">aoxb: 15,18-</span></p><p class="c3"><span class="c1 c4">rfp-f3: 1, 11, 12, 18, 19</span></p><p class="c3"><span class="c1 c4"> </span></p><p class="c3"><span class="c1 c2 c5">transformation of hyb-rfp-psb1a3 into bl21(Margo, Christina)</span></p><p class="c3"><span class="c1 c2 c5"> </span></p><p class="c3"><span class="c1">10 սL Nova Blue cells + 10 սL of ligation rxn</span></p><p class="c3"><span class="c1 c4 c5">See Protocols page for Heat Shock Transformation</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c5">to do(in addition to list)</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">make more lb +carb plates</span></li></ol><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">autoclave eppendorfs, toothpicks, tips</span></p><ol class=""><li class="c11" value="1"><span class="c1 c5">miniprep liq cultures of confirmed colonies (Margo)(done)</span></li></ol><p class="c3"><span class="c1 c5"> </span><span class="c1 c4 c5">See Protocols page for</span><span class="c1 c5"> </span><span class="c1 c4 c5">Plasmid DNA Purification with Mini Prep Kit</span></p><ol class=""><li class="c7" value="1"><span class="c1 c5">nanospec(margo done)</span></li></ol><ol class="c14"><li class="c11" value="2"><span class="c1 c5">TAKE OFF DOUBLE DIGESTS AT 4 PM (Heat block) (digests done today)</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1 c5">we put these back on to go overnight</span></li></ol><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c5">10/22/2010</span></p><p class="c3"><span class="c1 c2">goals</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">take pictures of the two plates labeled “hyb.rfp.psb1a3” before putting them into the 4c to track changes in colony color (Margo, Done)</span></li><li class="c11"><span class="c1">run digests from yesterday on gel - done (Christian, Margo)</span></li><li class="c11"><span class="c1">make lb+carb plates - done (Margo, Scott)</span></li><li class="c11"><span class="c1">using the transformations of the aoxa/b and rfp-f3r colonies as templates, resmear additional plates so that we can have distinct colonies as opposed to the lawns we have now - done (Margo)</span></li></ol><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">Protocols</span></p><p class="c3"><span class="c1 c2"> </span></p><p class="c3"><span class="c1 c2">Making LB media (Margo, Scott)</span></p><p class="c3"><span class="c1">To make 0.75 L LB (Lysogeny broth) medium:</span></p><p class="c3"><span class="c1">3.75 g Yeast extract</span></p><p class="c3"><span class="c1">7.5g Tryptone</span></p><p class="c3"><span class="c1">3.75g NaCl</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">To make solid plates, follow the above recipe and add 16g agar per 1L (12 for .75 l)</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Prepared 800 mL of LB media (50 ml extra)</span></p><p class="c3"><span class="c1">750 ml for plates and 250ml liquid</span></p><p class="c3"><span class="c1">Autoclave, leaving lids slightly loosened.</span></p><p class="c3"><span class="c1">Let cool in water bath at 55C. (started 12 pm)</span></p><p class="c3"><span class="c1">Add antibiotic (800uL carbenicillin) to 800 mL LB media used for plates.</span></p><p class="c3"><span class="c1">For the agar plates, pipetted 25mL into each plate, allow to hardened on benchtop. Refrigerated and ready to go..</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Note for others:</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">For resmearing the plates of the transformation done 10.21.2010 (aox 3, 5a, 6a, aox b15 and b18, rfp f3 1, 11, 12, 19). Make sure to save ALL plates when done and put the ones from 10.21.2010 back into the 4c fridge! We are tracking the progress of red colonies on the rfp-f3 paltes</span></li></ol><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Made liquid cultures of colonies A3, A5, and R11 from cryostocks; in incubator ~ 1:15 p.m. (Margo)</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Digest Gel</span></p><p class="c3"><span class="c1">(100bp) Ladder|1R|11R|12R|18R|19R|Ladder|A3|A5|A6|Ladder|B15|B18 </span><br/><img height="443.0" src="https://static.igem.org/mediawiki/2010/5/59/10-17.5.png" width="437.0"></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Made liquid cultures of colonies of hyBb-RFP-psB1a3 in BL21. I picked 6 colonies from plate 1. #6 was already red by the time I started the culture, the rest were pinkish. I also picked 7 colonies from plate 2. #12 and #13 were already red, and the rest were pinkish by the time I started the culture.</span></p><p class="c3"><span class="c1">The 13 tubes are in the 37 degrees incubator/ shaker in the other room. </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c5">How to prepares samples for microscopy (Look at table below)</span></p><p class="c3"><span class="c1">1. In cold, growing for a couple of days.</span></p><p class="c3"><span class="c1">Get a fresh plate...liquid of streaking of a colony.</span></p><p class="c3"><span class="c1">2 or several plates for cold temperature. 1 will be left at cold until the last minute. the other one should be moved to warm a couple hours before we go to the scope.</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">2 other plates... put them in 37 and grow them overnight... (saturday night let them grow), sunday transfer them to the cold, one of these will stay in the cold until the last minute, and one will be moved to warm a couple hours before we image.</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">another plate... at 37 the whole time.. never goes to cold. - do this on sunday preferably.</span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1">Once we have new plates that have been streaked with colonies from liquid culture on Saturday night, prepare the following samples: (I explaine this to Scott. If he’s not here, call him or me!)</span></p><table cellpadding="0" cellspacing="0" class="c16"><tbody><tr><td class="c0"><p class="c3"><span class="c1">Plate</span></p></td><td class="c0"><p class="c3"><span class="c1">When you have to move plates to new temperature</span></p></td><td class="c0"><p class="c3"><span class="c1">Conditions</span></p></td><td class="c13"><p class="c3"><span class="c1"> </span></p></td></tr><tr><td class="c0"><p class="c3"><span class="c1">1</span></p></td><td class="c0"><p class="c3"><span class="c1"> </span></p></td><td class="c0"><p class="c3"><span class="c1">Leave in fridge (4 degrees) until we image</span></p></td><td class="c13"><p class="c3"><span class="c1"> </span></p></td></tr><tr><td class="c0"><p class="c3"><span class="c1">2</span></p></td><td class="c0"><p class="c3"><span class="c1">Sunday night</span></p></td><td class="c0"><p class="c3"><span class="c1">Leave in 4 degrees until the last person leaves on Sunday night, put in 37 degrees incubator.</span></p></td><td class="c13"><p class="c3"><span class="c1"> </span></p></td></tr><tr><td class="c0"><p class="c3"><span class="c1">3</span></p></td><td class="c0"><p class="c3"><span class="c1">Monday morning</span></p></td><td class="c0"><p class="c3"><span class="c1">Leave in 4 degrees until Monday morning at 7 am, put in 37 degrees</span></p></td><td class="c13"><p class="c3"><span class="c1"> </span></p></td></tr></tbody></table><p class="c3"><span class="c1"> </span></p><table cellpadding="0" cellspacing="0" class="c16"><tbody><tr><td class="c0"><p class="c3"><span class="c1">4</span></p></td><td class="c0"><p class="c3"><span class="c1">Monday morning</span></p></td><td class="c0"><p class="c3"><span class="c1">Leave in 4 degrees until Monday morning at 8 am, put in 37 degrees</span></p></td><td class="c6"><p class="c3"><span class="c1"> </span></p></td></tr><tr><td class="c0"><p class="c3"><span class="c1">5</span></p></td><td class="c0"><p class="c3"><span class="c1">sunday day time</span></p></td><td class="c0"><p class="c3"><span class="c1">Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Leave in cold until we image</span></p></td><td class="c6"><p class="c3"><span class="c1"> </span></p></td></tr><tr><td class="c0"><p class="c3"><span class="c1">6</span></p></td><td class="c0"><p class="c3"><span class="c1">Sunday day time, sunday night</span></p></td><td class="c0"><p class="c3"><span class="c1">Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Move back to 37 sunday night before you leave.</span></p></td><td class="c6"><p class="c3"><span class="c1"> </span></p></td></tr><tr><td class="c0"><p class="c3"><span class="c1">7</span></p></td><td class="c0"><p class="c3"><span class="c1">Sunday night, monday morning</span></p></td><td class="c0"><p class="c3"><span class="c1">Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Move to 37 at 7 am on Monday morning</span></p></td><td class="c6"><p class="c3"><span class="c1"> </span></p></td></tr><tr><td class="c0"><p class="c3"><span class="c1">8</span></p></td><td class="c0"><p class="c3"><span class="c1">Sunday night, monday morning</span></p></td><td class="c0"><p class="c3"><span class="c1">Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Move to 37 at 8 am on Monday morning</span></p></td><td class="c6"><p class="c3"><span class="c1"> </span></p></td></tr><tr><td class="c0"><p class="c3"><span class="c1">9</span></p></td><td class="c0"><p class="c3"><span class="c1">never</span></p></td><td class="c0"><p class="c3"><span class="c1">Leave in 37 the whole time</span></p></td><td class="c6"><p class="c3"><span class="c1"> </span></p></td></tr></tbody></table><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c5">10/23/2010</span></p><p class="c3"><span class="c1 c5">goals (look at list in lab)</span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c2">protocols</span></p><ol class="c8"><li class="c7" value="1"><span class="c1">Digest of colony A5 of hyb.ompa.aoxA.psb1a3 (from 10.22.2010)(Margo)</span></li><li class="c7"><span class="c1 c5">colony 5, Aox1a colony PCR from 10-22 (117 ng/ul)</span></li><li class="c7"><span class="c1">4.5 uL H20</span></li><li class="c7"><span class="c1">2uL 10X EcoRI buffer</span></li><li class="c7"><span class="c1">10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.22.2010)</span></li><li class="c7"><span class="c1">2 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></li><li class="c7"><span class="c1">0.75 uL SpeI</span></li><li class="c7"><span class="c1">0.75 uL EcoRI</span></li><li class="c7"><span class="c1 c5">Total=20 ul total</span></li></ol><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c5">Note: we need SpeI or a equivalent enzyme with same cutting site and overhang! We couldn’t start the digest today!</span></p><p class="c3"><span class="c1 c5"> </span></p><p class="c3"><span class="c1 c2">observations</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">on the hyb-rfp plates from 10.22, we saw some colonies that were slightly pink that were not pink yesterday; they are circled and dated with today’s date.</span></li></ol><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1"> </span></p><p class="c3"><span class="c1 c2">heat characterization</span></p><ol class="c14"><li class="c11" value="1"><span class="c1">Making 3 l of lb and 1 l of lb+agar (Margo, Christian)</span></li><li class="c11"><span class="c1">we plated each of the 9 conditions (above table) using colony R11 liquid cultures from 10.22.2010</span></li></ol><ol class="c8"><li class="c7" value="1"><span class="c1">plates are in appropriate 4c or incubator conditions</span></li></ol><p class="c3"><span class="c1 c4"> </span></p> | |
Latest revision as of 02:57, 28 October 2010
10/17/2010
Goals
- transformations of NB of ligations done 10/16/2010
- hyb.ompa.aox
- hyb.ompa.aoxb
- hyb.ompa.rfp-f3r
- negative control
- miniprep of aoxb colony b5
See Protocols page for Plasmid DNA Purification with Mini Prep Kit
- nanospec the minipreps done 10/16/2010 and today
- check results of digests on gel
- digests of hyb.ompa.aoxa/b colonies b1-3, a10 with EcoRI and Spe I
- grow up a colony of psb1c3
Protocols
transformations of NB using ligations done 10/16/2010 (Christina)
- hyb.ompa.aox
- hyb.ompa.aoxb
- hyb.ompa.rfp-f3r
- negative control
10 ul of NB + 5 ul of ligation rxn
plate at 1 pm
gel of digests hyb.ompa.aoxa/b colonies b1-3, a10 with EcoRI and Spe I (Scott)
Note: problem with wells 2,3
order: ladder|b1|b2|b3|a10|b1|b2|ladder (exacto)
Colony aoxa A10 has the pattern we want: a 1 kb insert adn 2 kb bakbone
miniprep of colony b5 (Scott)
See Protocols page for Plasmid DNA Purification with Mini Prep Kit
nanospec
hyb.ompa.aox.psb1a3 colony b5 (10.16.2010)=90 ng/uL
hyb.ompa.aox.psb1a3 colony b5 (10.17.2010)=101 ng/uL
Repeat pcrs to make hyb-Fr, RFP-FR, aox1b FR, aox1a FR
Rxns
Hyb-F and Hyb-R (note use 1 ul of template for hyb)
RFP-F and RFP-R
Aox1b-F and Aox1b-R
aox1a-F and aox1a-R
PCR Protocol
26.5 uL H2O -
10 uL PHUSION 5X Reaction buffer -
5 uL forward primer -
5 uL reverse primer -
1 uL dNTP 10 mM - (thawed & kept on ice)
2 uL template DNA (note: use 1 uL for hybB) -
0.5 uL polymerase enzyme, PHUSION -
Total Volume= 50 uL
grow up a starter colony of psb1c3 (Christina)
The colonies were pinkish, so Christina picked a colony so we can miniprep them tomorrow
Digest of colony B5 of hyb.ompa.aoxb.psb1a3 (from 10.16.2010)(Margo)
colony 5, Aox1b colony PCR from 10-16: Label on tube in freezer is 516
4.5 uL H20
2uL 10X EcoRI buffer
10 uL hyb.ompa.aoxb.psb1a3 colony 5 miniprep (10.16.2010)
2 uL BSA (1ug/uL, to a final conc of .1mg/ml)
0.75 uL SpeI
0.75 uL EcoRI
Total=20 ul total
colony 5, Aox1b colony PCR from 10-17: Label on tube in freezer is 517
4.5 uL H20
2uL 10X EcoRI buffer
10 uL hyb.ompa.aoxb.psb1a3 colony 5 miniprep (10.17.2010)
2 uL BSA (1ug/uL, to a final conc of .1mg/ml)
0.75 uL SpeI
0.75 uL EcoRI
Total=20 ul total
Into heat block at 1:51 p.m., remove at 4:51 p.m.
Making Gels
Started at 2:26 pm. Gels ready at 3:26 pm.
Gels:
- 4 ul of the PCRs Christina did today
- aoxa FR
- aoxb FR
- hyb FR
- RFp FR
- RE digest 516
- RE digest 517
Gel started 5:00 pm.
Results:
10/18/2010
Goals
- Send off for sequencing the minirep of colony a10
Colony PCR of hyb.ompa+aoxa+psb1a3, hyb.ompa+aoxb+psb1a3, hyb.ompa+RFP-F3R+psb1a3
2.5 uL of cells
11.75 uL H2O
5 uL GOTAQ 5X Reaction buffer
2.5 uL Hyb-F forward primer
2.5 uL Ompa-R reverse primer
.5 uL dNTP 10 mM - (thawed & kept on ice)
0.25 uL polymerase enzyme, TAQ
Total Volume= 25 uL
Picked four white colonies from each plate, added to 25uL water.
Plate | Microcentrifuge Tube Numbers |
psB1A3-aox1A (negative control, 10/17) | 1,2,3,4 |
psB1A3-aox1B-hybB-ompA (10/17) | 5,6,7,8 |
psB1A3-aox1A-hybB-ompA (10/17) | 9,10,11,12 |
psB1A3-hybB-ompA-RFP,F3R (10/17) | 13,14,15,16 |
Run gel on colony PCRS from above.
Lanes were run in order (1-16), with a 1KB ladder middle band (there was only 2uL of this left, so the ladder is faint).
Gel electrophoresis of Top and Bottom bands from 17 OCT 2010
Ladder | Top | Bottom (yes, both are very faint)
Ladder | Top | Bottom
Retransformation of Ligations from 10-16
Repeated heat shock transformations of ligations from 10-16-10.
Used 5uL of ligation reaction + 10uL of NB cells.
Plated 100uL of each on each plate.
See Protocols page for Heat Shock Transformation
10/19/2010
Goals
- reload colony pcrs from 10/18/2010 if need be (the ladder is faint, so if no interpretation is possible, then load again. otherwise no need to)
- Load digest the 2 digests of colony B5 hyb.ompa.aoxb.psb1a3 from 10.17.2010 (done)
- if they have the expected insert, send off for sequencing
- Load the pcrs from 10.17 of hyb-FR, aoxa-Fr, aoxb-FR, and rfp-F3R on a gel (done)
- pick (20) colonies of RFP-F3 from 10/18/2010 (done)
- after the colony pcr, add 3ml of LB instead of 250 ul. This will prepare us for minipreps tomorrow.
- Pick (20) colonies of aoxb (from 10.18/2010 plates) (done)
- after the colony pcr, add 3ml of LB instead of 250 ul. This will prepare us for minipreps tomorrow.
- try the ligation of hyb-rfp-psb1a3 , transform into NB or bl21
- transform the “sequenced” colonies (a10) into bl21
- miniprep of psb1C3 (done)
- Send colony a10 for sequencing (Christina)(to be done)
- grow up some more colony a10 from glycerol stock
Protocols
loading gel (done, Scott)
order:
exact ladder|aoxa-FR phusion pcr (10.17.2010)||aoxb-FR phusion pcr (10.17.2010)||hyb-FR phusion pcr (10.17.2010)||RFP-F3R phusion pcr (10.17.2010)|exact ladder| digest of miniprep of colony b5 (10.16 version) hyb.omp.aoxb.psb1a3 from 10.17.2010|digest of miniprep of colony b5 (10.17 version) hyb.omp.aoxb.psb1a3 from 10.17.2010
start 11:08 am
the pcrs appeared to work, but the digests do not contain the predicted band pattern.
Miniprep of psb1C3(Scott) (done)
See Protocols page for Plasmid DNA Purification with Mini Prep Kit
Prepare DNA to send off for sequencing.( Christina)(Scott)
In standard PCR tubes, prepare template DNA with just water and ONE primer (forward or reverse). The total volume should be 15 uL. The concentration of DNA in the sample should be adjusted to match the length of the template DNA; there is a chart in the Gaucher lab on the wall above the EtBr disposal bucket. For example, for a PCR product 1000 to 2000 bp in length, the concentration of the sample should be ~ 4 ng/uL.
Predicted size of hybB-ompA-aox1a: 1509 bp. Calculate concentration to be ~ 4 ng/uL
An email should be sent to Ryan with the following information:
Tube Label: actually written on the tube, in this form: RR01, RR02, etc.
DNA Name: this is something meaningful to us, like AOXa_construct_PSB1A3_vector_1
DNA Type: Purified PCR or Plasmid
DNA length: in bp
My primer: a name meaningful to us
Give the tubes to Ryan. Apparently they should not be frozen, just kept on ice.
Rxns: colony 10 hyb.ompa.aoxa miniprep from 10/15/2010
psb1a3-F
hybF
aox-F
aox-middle
colony PCRs (Rob, Christian, Scott)
use plates from 10/18/2010
use hyb-f and ompa-r
Reactions:
aoxa 1-20
aoxb 1-20
rfp-f3r 1-20
Colony pcr Master mix (batch of 70 amount in brackets)
11.75 uL H2O (822.5 uL)-
5 uL GOTAQ 5X Reaction buffer (350 uL)-
2.5 uL Hyb-F forward primer (175 uL)-
2.5 uL Ompa-R reverse primer (175 uL)-
.5 uL dNTP 10 mM - (thawed & kept on ice) (35 uL)-
0.25 uL polymerase enzyme, TAQ (17.5 uL)
2.5 uL of cells added to each respective PCR tube
Total Volume= 25 uL
Start: 3:31 pm
10/20/2010
goals
- load rest of colony pcrs on gel from 10/19
- load 5 ul
protocols
loading gels for colony pcrs (Scott)
expected fragments:
hyb-f and ompa-r approx 500 bp.
gel one (started 9:36 am)
order:
exact ladder|aoxa colony pcrs 1-6|ladder|aoxa colony pcrs 7-14|ladder
gel two
exact ladder|aoxa colony pcrs 15-20|colony pcr aoxb 1|ladder|two empty wells|ladder|aoxb colony pcrs 2-6|
gel three
order
exact ladder|aoxb colony pcrs 7-12 (from 10.19.2010)|ladder
gel four
order
exact ladder|aoxb colony pcrs 13-18 (from 10.19.2010)|ladder
(gel 3 and 4 are together below)
gel five
order
exact ladder|aoxb colony pcrs 19-20 (from 10.19.2010)|rfp-f3r colonies 1-4ladder
gel six
order
exact ladder|rfp-f3r colony pcrs 5-10(from 10.19.2010)|ladder
(gel 5 and 6 are below)
gel seven
order
exact ladder|rfp-f3r colony pcrs 10-20(from 10.19.2010)|ladder
RE Double Digest Recipe for pSB1C3 miniprep (from 10/19/2010) -- digest with speI and EcorI (Debika)
STARTED AT 2.54 PM
12.5 uL H20 -
3uL EcoRI Buffer -
10 uL pSB1C3 (10.8.2010, 56 ng/uL)
3 uL BSA (1ug/uL, to a final conc of .1mg/ml) -
0.75 uL SpeI
0.75 uL EcoRI
Total=30 ul total
Digest overnight - put in waterbath 37 degrees at 2.54 PM
Predict a insert (1000 bp) an(?- this was already there from the last protocol that Scott copied for me, DM)
Things to do
1. load colonies rfp-f3r 10-20 on gel, visualize for 500 bp band (done Christina)
2. colony pcr using aoxa/b f-R from colonies on 10/19/2010 (Done Christina)
3. grow up good colonies in 5ml liq culture
colony pcr using aoxa/b f-R from colonies on 10/19/2010
Do 2nd pcr on colonies (from 10/19/2010)
aoxa: 3,5,6
aoxb: 15,18
rfp-f3: 1, 11, 12, 18, 19
Note: change the colony pcr program to have an extension time of 1.5 mins.
For aoxa (colonies 3, 5,6)
11.75 uL H2O
5 uL GOTAQ 5X Reaction buffer
2.5 uL Hyb F forward primer
2.5 uL Aoxa R reverse primer
.5 uL dNTP 10 mM - (thawed & kept on ice)
0.25 uL polymerase enzyme, TAQ
.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube
Total Volume= 25 uL
For aoxb (colonies 15, 18)
11.75 uL H2O
5 uL GOTAQ 5X Reaction buffer
2.5 uL Hyb F forward primer
2.5 uL Aoxb R reverse primer
.5 uL dNTP 10 mM - (thawed & kept on ice)
0.25 uL polymerase enzyme, TAQ
.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube
Total Volume= 25 uL
For RFP f3r (colonies 1, 11, 12, 18, 19)
11.75 uL H2O
5 uL GOTAQ 5X Reaction buffer
2.5 uL HybF forward primer
2.5 uL Rfp-r reverse primer
.5 uL dNTP 10 mM - (thawed & kept on ice)
0.25 uL polymerase enzyme, TAQ
.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube
Total Volume= 25 uL
*NOTE - can use the PCR reaction (.25uL) from today on aoxa 3,5,6; aoxb 15, 18; rfp-f3 1 as template DNA. (good point)
Ligation of RFP-FR,HybB-FR,to psb1a3 (Christina)
Calculating equivalents: 9C
rfp (from9.22.2010)
psb1a3 (10.15, top )
hyb (from 9.10, purified 10.20)
RFP- [41ng/uL]/678 bp= 0.06eq/uL
hyBb-[ 6 ng/uL]/393 bp = 0.0152 eq/uL
psb1a3-[ 9.4 ng/ul]/2000 bp= 0.0047 eq/uL
for linear ligations, use a 2:2:1 of insert:insert:vector
0.5 uL of RFP FR
2 uL of HybB FR
3 ul psb1a3
1 uL 10x Ligase Buffer
3 uL H20
0.5 uL T4 Ligase
Total=10 uL
overnight 4c
for tomorrow
- look on list for any overlaps and missed items
- note: make glycerol stocks when doing minipreps
10/21/2010
goals
- glycerol stocks of liquid cultures (done)
- repeat pcr from 10/20 checking the “good” colonies; the gel showed no predicted bands, but that may be due to pcr errors, elongation time etc. Try using aoxa/b f-r only, or try troubleshooting why the pcr did not make the 1.5 kb band
- miniprep liq cultures of confirmed colonies (Scott)(done)
- nanospec’ed
- send ones that are confirmed for sequencing
- digest the three inserts from 3 good colonies and ligate to psb1c3
- make sure to pcr purify the digest from yesrerday
- transform bl21 using hyb-rfp ligation
- transform bl21 using minipreps of good colonies
- check list for anything else!
protocols
glycerol stocks of liq cultures(Christian)
colonies:
aoxa: 3,5,6
aoxb: 15,18
rfp-f3: 1, 11, 12, 18, 19
loading gel to check pcrs yesterday (Scott)
rxns:
colony pcr using aoxa/b f-R from colonies on 10/19/2010
aoxa using hyb-f and aoxa-r: 3,5,6
aoxb using hyb-f and aoxb-r: 15,18
rfp-f3 using hyb-f and rfp-r: 1, 11, 12, 18, 19 (used hyb-f and rfp-r)
order
exact ladder|aoxa colony 3,5,6|aoxb colony 15,18,|rfp-f3r colony1|ladder|rfp-f3r colony 11,12,18,19|ladder
version 2 of above gel:
RE Double Digest Recipe miniprep (from 10/21/2010) -- digest with speI and EcoRI
12.5 uL H20 -
3uL EcoRI Buffer -
5 uL miniprep -
3 uL BSA (1ug/uL, to a final conc of .1mg/ml) -
0.75 uL SpeI
0.75 uL EcoRI
Total=30 ul total
3 hour digest
started 1pm
predicted sizes:
2kb (backbone)
hyb.ompa./aoxa/b insert approx 1kb
pcr using aoxa/b FR (done)
rxns
aoxa using aoxa-f and aoxa-r: 3,5,6-
aoxb using aoxb-f and aoxb-r: 15,18-
rfp-f3 using rfp-f and rfp-r: 1, 11, 12, 18, 19- (used hyb-f and rfp-r)
- 26.5 uL H2O-
- 10 uL Taq 5X Reaction buffer-
- 5 uL forward primer-
- 5 uL reverse primer-
- 1 uL dNTP 10 mM - (thawed & kept on ice)-
- 2 uL template DNA-
- 0.5 uL polymerase enzyme, Gotaq
Total Volume= 50 uL
started 1:30pm
prepare DNA to send off for sequencing.( Margo)
In standard PCR tubes, prepare template DNA with just water and ONE primer (forward or reverse). The total volume should be 15 uL. The concentration of DNA in the sample should be adjusted to match the length of the template DNA; there is a chart in the Gaucher lab on the wall above the EtBr disposal bucket. For example, for a PCR product 1000 to 2000 bp in length, the concentration of the sample should be ~ 4 ng/uL.
Predicted size of hybB-ompA-aox1a: 1509 bp. Calculate concentration to be (check sheet)
An email should be sent to Ryan with the following information:
Tube Label: actually written on the tube, in this form: RR01, RR02, etc.
DNA Name: this is something meaningful to us, like AOXa_construct_PSB1A3_vector_1
DNA Type: Purified PCR or Plasmid
DNA length: in bp
My primer: a name meaningful to us
Give the tubes to Ryan. Apparently they should not be frozen, just kept on ice.
Rxns: colony aox5(a5) , aoxb 18(18b), rfp-f3r colony 1 (1r)
psb1a3-F
hybF
aox-F
aox-middle
RR01 = Aox1a colony A5, with psb1a3 forward primer
RR02 = Aox1a colony A5, with AOX1a forward primer
RR03 = Aox1a colony A5, with AOX1a middle forward primer
RR04 = Aox1a colony B18, with psb1a3 forward primer
RR05 = Aox1a colony B18, with AOX1b forward primer
RR06 = Aox1a colony B18, with AOX1b middle forward primer
RR07 = RFP colony R12, with psb1a3 forward primer
RR08 = RFP colony R12, with RFP3 forward primer
RR01 AOX1a_colonyA5_psb1a3_primer 1500 bp psb1A3_forward
RR02 AOX1a_colonyA5_aox1af_primer 1500 bp aox1a_forward
RR03 AOX1a_colonyA5_aox1amid_primer 1500 bp aox1amid_forward
RR04 AOX1b_colonyB18_psb1a3_primer 1500 bp psb1A3_forward
RR05 AOX1b_colonyB18_aox1bf_primer 1500 bp aox1b_forward
RR06 AOX1b_colonyB18_aox1bmid_primer 1500 bp aox1bmid_forward
RR07 RFP_colonyR12_psb1a3_primer 1200 bp psb1A3_forward
RR08 RFP_colonyR12_RFP3_primer 1200 bp RFP3_forward
aoxa using aoxa-f and aoxa-r: 3,5,6-
aoxb using aoxb-f and aoxb-r: 15,18-
rfp-f3 using rfp-f and rfp-r: 1, 11, 12, 18, 19- (used hyb-f and rfp-r)
loading gel
gel 1 and 2 are of digests using ecoRI and SpeO
Gel 1(left)
ladder|A3|A5|A6|B15|B18|empty|ladder
Gel 2(right)
ladder|R1|R11|R12|R18|R19|empty|ladder
gel 3
PCR from 10.21.2010 using aoxa/b-FR or rfp-FR (for Rfp-f3r constructs)
of colonies from hyb.ompa.aoxa/b or rfp-f3r colonies
order
ladder|aox 3a|aox5a|aox 6a|aox 15b|aox 18b|rfp-f3r 1R|ladder| rfp-f3r 11R| rfp-f3r 19R|rfp-f3r18r|ladder
transformations into bl21: (Christina, Christian)
aoxa: 3,5,6-
aoxb: 15,18-
rfp-f3: 1, 11, 12, 18, 19
transformation of hyb-rfp-psb1a3 into bl21(Margo, Christina)
10 սL Nova Blue cells + 10 սL of ligation rxn
See Protocols page for Heat Shock Transformation
to do(in addition to list)
- make more lb +carb plates
autoclave eppendorfs, toothpicks, tips
- miniprep liq cultures of confirmed colonies (Margo)(done)
See Protocols page for Plasmid DNA Purification with Mini Prep Kit
- nanospec(margo done)
- TAKE OFF DOUBLE DIGESTS AT 4 PM (Heat block) (digests done today)
- we put these back on to go overnight
10/22/2010
goals
- take pictures of the two plates labeled “hyb.rfp.psb1a3” before putting them into the 4c to track changes in colony color (Margo, Done)
- run digests from yesterday on gel - done (Christian, Margo)
- make lb+carb plates - done (Margo, Scott)
- using the transformations of the aoxa/b and rfp-f3r colonies as templates, resmear additional plates so that we can have distinct colonies as opposed to the lawns we have now - done (Margo)
Protocols
Making LB media (Margo, Scott)
To make 0.75 L LB (Lysogeny broth) medium:
3.75 g Yeast extract
7.5g Tryptone
3.75g NaCl
To make solid plates, follow the above recipe and add 16g agar per 1L (12 for .75 l)
Prepared 800 mL of LB media (50 ml extra)
750 ml for plates and 250ml liquid
Autoclave, leaving lids slightly loosened.
Let cool in water bath at 55C. (started 12 pm)
Add antibiotic (800uL carbenicillin) to 800 mL LB media used for plates.
For the agar plates, pipetted 25mL into each plate, allow to hardened on benchtop. Refrigerated and ready to go..
Note for others:
- For resmearing the plates of the transformation done 10.21.2010 (aox 3, 5a, 6a, aox b15 and b18, rfp f3 1, 11, 12, 19). Make sure to save ALL plates when done and put the ones from 10.21.2010 back into the 4c fridge! We are tracking the progress of red colonies on the rfp-f3 paltes
Made liquid cultures of colonies A3, A5, and R11 from cryostocks; in incubator ~ 1:15 p.m. (Margo)
Digest Gel
(100bp) Ladder|1R|11R|12R|18R|19R|Ladder|A3|A5|A6|Ladder|B15|B18 
Made liquid cultures of colonies of hyBb-RFP-psB1a3 in BL21. I picked 6 colonies from plate 1. #6 was already red by the time I started the culture, the rest were pinkish. I also picked 7 colonies from plate 2. #12 and #13 were already red, and the rest were pinkish by the time I started the culture.
The 13 tubes are in the 37 degrees incubator/ shaker in the other room.
How to prepares samples for microscopy (Look at table below)
1. In cold, growing for a couple of days.
Get a fresh plate...liquid of streaking of a colony.
2 or several plates for cold temperature. 1 will be left at cold until the last minute. the other one should be moved to warm a couple hours before we go to the scope.
2 other plates... put them in 37 and grow them overnight... (saturday night let them grow), sunday transfer them to the cold, one of these will stay in the cold until the last minute, and one will be moved to warm a couple hours before we image.
another plate... at 37 the whole time.. never goes to cold. - do this on sunday preferably.
Once we have new plates that have been streaked with colonies from liquid culture on Saturday night, prepare the following samples: (I explaine this to Scott. If he’s not here, call him or me!)
Plate | When you have to move plates to new temperature | Conditions |
|
1 |
| Leave in fridge (4 degrees) until we image |
|
2 | Sunday night | Leave in 4 degrees until the last person leaves on Sunday night, put in 37 degrees incubator. |
|
3 | Monday morning | Leave in 4 degrees until Monday morning at 7 am, put in 37 degrees |
|
4 | Monday morning | Leave in 4 degrees until Monday morning at 8 am, put in 37 degrees |
|
5 | sunday day time | Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Leave in cold until we image |
|
6 | Sunday day time, sunday night | Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Move back to 37 sunday night before you leave. |
|
7 | Sunday night, monday morning | Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Move to 37 at 7 am on Monday morning |
|
8 | Sunday night, monday morning | Leave in 37 degrees overnight (Saturday), move to cold on Sunday (daytime). Move to 37 at 8 am on Monday morning |
|
9 | never | Leave in 37 the whole time |
|
10/23/2010
goals (look at list in lab)
protocols
- Digest of colony A5 of hyb.ompa.aoxA.psb1a3 (from 10.22.2010)(Margo)
- colony 5, Aox1a colony PCR from 10-22 (117 ng/ul)
- 4.5 uL H20
- 2uL 10X EcoRI buffer
- 10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.22.2010)
- 2 uL BSA (1ug/uL, to a final conc of .1mg/ml)
- 0.75 uL SpeI
- 0.75 uL EcoRI
- Total=20 ul total
Note: we need SpeI or a equivalent enzyme with same cutting site and overhang! We couldn’t start the digest today!
observations
- on the hyb-rfp plates from 10.22, we saw some colonies that were slightly pink that were not pink yesterday; they are circled and dated with today’s date.
heat characterization
- Making 3 l of lb and 1 l of lb+agar (Margo, Christian)
- we plated each of the 9 conditions (above table) using colony R11 liquid cultures from 10.22.2010
- plates are in appropriate 4c or incubator conditions
