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| + | <title>10/17-10/23</title><style type="text/css">ol{margin:0;padding:0}p{margin:0}.c11{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}.c7{vertical-align:sub;color:#ffffff;font-size:6.666666666666666pt;font-family:Arial}.c8{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c12{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:72.0pt}.c2{line-height:1.15;text-indent:0pt;direction:ltr}.c0{color:#ffffff;font-size:10pt;font-family:Arial}.c9{line-height:1.0;text-indent:0pt;direction:ltr}.c13{background-color:#ffffff}.c3{list-style-type:circle}.c10{font-weight:bold}.c4{font-style:italic}.c5{border-collapse:collapse}.c1{text-decoration:underline}.c6{list-style-type:disc}</style></head><body class="c13"><p class="c2"><span class="c0 c10">10/17/2010</span></p><p class="c2"><span class="c0 c1">Goals</span></p><ol class=""><li class="c8" value="1"><span class="c0">transformations of NB of ligations done 10/16/2010</span></li></ol><ol class=""><li class="c12" value="1"><span class="c0">hyb.ompa.aox</span></li><li class="c12"><span class="c0">hyb.ompa.aoxb</span></li><li class="c12"><span class="c0">hyb.ompa.rfp-f3r</span></li><li class="c12"><span class="c0">negative control</span></li></ol><ol class="c6"><li class="c8" value="2"><span class="c0">miniprep of aoxb colony b5 </span></li><li class="c8"><span class="c0">nanospec the minipreps done 10/16/2010 and today</span></li><li class="c8"><span class="c0">check results of digests on gel</span></li></ol><ol class="c3"><li class="c12" value="1"><span class="c0">digests of hyb.ompa.aoxa/b colonies b1-3, a10 with EcoRI and Spe I</span></li></ol><ol class=""><li class="c8" value="5"><span class="c0">grow up a colony of psb1c3</span></li></ol><p class="c2"><span class="c0 c10 c1"> </span></p><p class="c2"><span class="c0 c1">Protocols</span></p><p class="c2"><span class="c0 c1"> </span></p><p class="c2"><span class="c0 c1">transformations of NB using ligations done 10/16/2010 (Christina)</span></p><ol class=""><li class="c12" value="1"><span class="c0">hyb.ompa.aox</span></li><li class="c12"><span class="c0">hyb.ompa.aoxb</span></li><li class="c12"><span class="c0">hyb.ompa.rfp-f3r</span></li><li class="c12"><span class="c0">negative control</span></li></ol><p class="c2"><span class="c0">10 ul of NB + 5 ul of ligation rxn</span></p><p class="c2"><span class="c0">plate at 1 pm </span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">gel of digests hyb.ompa.aoxa/b colonies b1-3, a10 with EcoRI and Spe I (Scott)</span></p><p class="c2"><span class="c0">Note: problem with wells 2,3</span></p><p class="c2"><span class="c0 c4">order: ladder|b1|b2|b3|a10|b1|b2|ladder (exacto)</span></p><p class="c2"><span class="c0">Colony aoxa A10 has the pattern we want: a 1 kb insert adn 2 kb bakbone</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">miniprep of colony b5 (Scott)</span></p><p class="c2"><span class="c0 c1"> </span></p><p class="c2"><span class="c0 c1">nanospec</span></p><p class="c2"><span class="c0">hyb.ompa.aox.psb1a3 colony b5 (10.16.2010)=90 ng/uL</span></p><p class="c2"><span class="c0">hyb.ompa.aox.psb1a3 colony b5 (10.17.2010)=101 ng/uL</span></p><p class="c2"><span class="c0 c1"> </span></p><p class="c2"><span class="c0 c1">Repeat pcrs to make hyb-Fr, RFP-FR, aox1b FR, aox1a FR</span></p><p class="c2"><span class="c0 c10">Rxns </span></p><p class="c2"><span class="c0 c10">Hyb-F and Hyb-R (note use 1 ul of template for hyb)</span></p><p class="c2"><span class="c0 c10">RFP-F and RFP-R</span></p><p class="c2"><span class="c0 c10">Aox1b-F and Aox1b-R</span></p><p class="c2"><span class="c0 c10">aox1a-F and aox1a-R</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">PCR Protocol </span><span class="c0"> </span></p><p class="c2"><span class="c0">26.5 uL H2O -</span></p><p class="c2"><span class="c0">10 uL </span><span class="c0 c10">PHUSION 5X Reaction buffer -</span></p><p class="c2"><span class="c0">5 uL forward primer -</span></p><p class="c2"><span class="c0">5 uL reverse primer -</span></p><p class="c2"><span class="c0">1 uL dNTP 10 mM - (thawed & kept on ice)</span></p><p class="c2"><span class="c0 c10">2 uL</span><span class="c0"> template DNA (note: use 1 uL for hybB) - </span></p><p class="c2"><span class="c0">0.5 uL polymerase enzyme, </span><span class="c0 c10">PHUSION -</span></p><p class="c2"><span class="c0 c10">Total Volume= 50 uL</span></p><p class="c2"><span class="c0 c1"> </span></p><p class="c2"><span class="c0 c1">grow up a starter colony of psb1c3 (Christina)</span></p><p class="c2"><span class="c0">The colonies were pinkish, so Christina picked a colony so we can miniprep them tomorrow</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">Digest of colony B5 of hyb.ompa.aoxb.psb1a3 (from 10.16.2010)(Margo)</span></p><p class="c2"><span class="c0 c10">colony 5, Aox1b colony PCR from 10-16: Label on tube in freezer is 5</span><span class="c7 c10">16</span></p><p class="c2"><span class="c0">4.5 uL H20 </span></p><p class="c2"><span class="c0">2uL 10X EcoRI buffer</span></p><p class="c2"><span class="c0">10 uL hyb.ompa.aoxb.psb1a3 colony 5 miniprep (10.16.2010)</span></p><p class="c2"><span class="c0">2 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c2"><span class="c0">0.75 uL SpeI</span></p><p class="c2"><span class="c0">0.75 uL EcoRI</span></p><p class="c2"><span class="c0 c10">Total=20 ul total </span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0 c10">colony 5, Aox1b colony PCR from 10-17: Label on tube in freezer is 5</span><span class="c7 c10">17</span></p><p class="c2"><span class="c0">4.5 uL H20 </span></p><p class="c2"><span class="c0">2uL 10X EcoRI buffer</span></p><p class="c2"><span class="c0">10 uL hyb.ompa.aoxb.psb1a3 colony 5 miniprep (10.17.2010)</span></p><p class="c2"><span class="c0">2 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c2"><span class="c0">0.75 uL SpeI</span></p><p class="c2"><span class="c0">0.75 uL EcoRI</span></p><p class="c2"><span class="c0 c10">Total=20 ul total </span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0 c10">Into heat block at 1:51 p.m., remove at 4:51 p.m.</span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0 c1">Making Gels</span></p><p class="c2"><span class="c0">Started at 2:26 pm. Gels ready at 3:26 pm. </span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0">Gels:</span></p><ol class="c6"><li class="c8" value="1"><span class="c0">4 ul of the PCRs Christina did today </span></li></ol><ol class="c3"><li class="c12" value="1"><span class="c0">aoxa FR</span></li><li class="c12"><span class="c0">aoxb FR</span></li><li class="c12"><span class="c0">hyb FR</span></li><li class="c12"><span class="c0">RFp FR</span></li></ol><ol class=""><li class="c8" value="2"><span class="c0">RE digest 5</span><span class="c7">16</span></li><li class="c8"><span class="c0">RE digest 5</span><span class="c7">17</span></li></ol><p class="c2"><span class="c0">Gel started 5:00 pm. </span></p><p class="c2"><span class="c0">Results: </span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c10">10/18/2010</span></p><p class="c2"><span class="c0 c1">Goals</span></p><ol class="c6"><li class="c8" value="1"><span class="c0">Send off for sequencing the minirep of colony a10 </span></li></ol><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">Colony PCR of hyb.ompa+aoxa+psb1a3, hyb.ompa+aoxb+psb1a3, hyb.ompa+RFP-F3R+psb1a3</span></p><p class="c2"><span class="c0">2.5 uL of cells</span></p><p class="c2"><span class="c0">11.75 uL H2O</span></p><p class="c2"><span class="c0">5 uL GOTAQ</span><span class="c0 c10"> 5X Reaction buffer</span></p><p class="c2"><span class="c0">2.5 uL Hyb-F forward primer</span></p><p class="c2"><span class="c0">2.5 uL Ompa-R reverse primer</span></p><p class="c2"><span class="c0">.5 uL dNTP 10 mM - (thawed & kept on ice)</span></p><p class="c2"><span class="c0">0.25 uL polymerase enzyme, TAQ</span></p><p class="c2"><span class="c0 c10">Total Volume= 25 uL </span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0">Picked four white colonies from each plate, added to 25uL water. </span></p><p class="c2"><span class="c0"> </span></p><table cellpadding="0" cellspacing="0" class="c5"><tbody><tr><td class="c11"><p class="c9"><span class="c0 c10">Plate</span></p></td><td class="c11"><p class="c9"><span class="c0 c10">Microcentrifuge Tube Numbers</span></p></td></tr><tr><td class="c11"><p class="c9"><span class="c0">psB1A3-aox1A (negative control, 10/17)</span></p></td><td class="c11"><p class="c9"><span class="c0">1,2,3,4</span></p></td></tr><tr><td class="c11"><p class="c9"><span class="c0">psB1A3-aox1B-hybB-ompA (10/17)</span></p></td><td class="c11"><p class="c9"><span class="c0">5,6,7,8</span></p></td></tr><tr><td class="c11"><p class="c9"><span class="c0">psB1A3-aox1A-hybB-ompA (10/17)</span></p></td><td class="c11"><p class="c9"><span class="c0">9,10,11,12</span></p></td></tr><tr><td class="c11"><p class="c9"><span class="c0">psB1A3-hybB-ompA-RFP,F3R (10/17)</span></p></td><td class="c11"><p class="c9"><span class="c0">13,14,15,16</span></p></td></tr></tbody></table><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">Run gel on colony PCRS from above. </span></p><p class="c2"><span class="c0">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="c2"><img height="448.0" src="images/image0.png" width="598.0"></p><p class="c2"><span class="c0 c1"> </span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">Gel electrophoresis of Top and Bottom bands from 17 OCT 2010</span></p><p class="c2"><img height="0.0" src="images/" width="0.0"></p><p class="c2"><img height="504.0" src="images/image7.png" width="674.0"></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0">Ladder | Top | Bottom (yes, both are very faint)</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><img height="520.0" src="images/image5.png" width="696.0"></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0">Ladder | Top | Bottom</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">Retransformation of Ligations from 10-16</span></p><p class="c2"><span class="c0">Repeated heat shock transformations of ligations from 10-16-10. </span></p><p class="c2"><span class="c0">Used 5uL of ligation reaction + 10uL of NB cells. </span></p><p class="c2"><span class="c0">Plated 100uL of each on each plate.</span></p><p class="c2"><span class="c0 c10 c4">See Protocols page for Heat Shock Transformation</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c10">10/19/2010</span></p><p class="c2"><span class="c0 c1">Goals</span></p><ol class="c6"><li class="c8" value="1"><span class="c0">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="c8"><span class="c0">Load digest the 2 digests of colony B5 hyb.ompa.aoxb.psb1a3 from 10.17.2010 (done)</span></li></ol><ol class="c3"><li class="c12" value="1"><span class="c0">if they have the expected insert, send off for sequencing</span></li></ol><ol class="c6"><li class="c8" value="3"><span class="c0">Load the pcrs from 10.17 of hyb-FR, aoxa-Fr, aoxb-FR, and rfp-F3R on a gel (done)</span></li><li class="c8"><span class="c0">pick (20) colonies of RFP-F3 from 10/18/2010 (done)</span></li></ol><ol class="c3"><li class="c12" value="1"><span class="c0">after the colony pcr, add 3ml of LB instead of 250 ul. This will prepare us for minipreps tomorrow.</span></li></ol><ol class="c6"><li class="c8" value="5"><span class="c0">Pick (20) colonies of aoxb (from 10.18/2010 plates) (done)</span></li></ol><ol class="c3"><li class="c12" value="1"><span class="c0">after the colony pcr, add 3ml of LB instead of 250 ul. This will prepare us for minipreps tomorrow.</span></li></ol><ol class="c6"><li class="c8" value="6"><span class="c0">try the ligation of hyb-rfp-psb1a3 , transform into NB or bl21</span></li><li class="c8"><span class="c0">transform the “sequenced” colonies (a10) into bl21</span></li><li class="c8"><span class="c0">miniprep of psb1C3 (done)</span></li><li class="c8"><span class="c0">Send colony a10 for sequencing (Christina)(to be done)</span></li><li class="c8"><span class="c0">grow up some more colony a10 from glycerol stock</span></li></ol><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">Protocols</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">loading gel (done, Scott)</span></p><p class="c2"><span class="c0 c4">order:</span></p><p class="c2"><span class="c0 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="c2"><span class="c0 c4">start 11:08 am</span></p><p class="c2"><span class="c0">the pcrs appeared to work, but the digests do not contain the predicted band pattern.</span></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c1">Miniprep of psb1C3(Scott) (done)</span></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c1">prepare DNA to send off for sequencing.( Christina)(Scott)</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0">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="c2"><span class="c0 c10">Predicted size of hybB-ompA-aox1a: 1509 bp. Calculate concentration to be ~ 4 ng/uL</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0">An email should be sent to Ryan with the following information:</span></p><p class="c2"><span class="c0">Tube Label: actually written on the tube, in this form: RR01, RR02, etc.</span></p><p class="c2"><span class="c0">DNA Name: this is something meaningful to us, like AOXa_construct_PSB1A3_vector_1</span></p><p class="c2"><span class="c0">DNA Type: Purified PCR or Plasmid</span></p><p class="c2"><span class="c0">DNA length: in bp</span></p><p class="c2"><span class="c0">My primer: a name meaningful to us</span></p><p class="c2"><span class="c0">Give the tubes to Ryan. Apparently they should not be frozen, just kept on ice.</span></p><p class="c2"><span class="c0 c4">Rxns: colony 10 hyb.ompa.aoxa miniprep from 10/15/2010</span></p><p class="c2"><span class="c0 c4">psb1a3-F</span></p><p class="c2"><span class="c0 c4">hybF</span></p><p class="c2"><span class="c0 c4">aox-F</span></p><p class="c2"><span class="c0 c4">aox-middle</span></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c1">colony PCRs (Rob, Christian, Scott)</span></p><p class="c2"><span class="c0">use plates from 10/18/2010</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c4">use hyb-f and ompa-r</span></p><p class="c2"><span class="c0 c4">Reactions:</span></p><p class="c2"><span class="c0">aoxa 1-20</span></p><p class="c2"><span class="c0">aoxb 1-20</span></p><p class="c2"><span class="c0">rfp-f3r 1-20</span></p><p class="c2"><span class="c0 c1"> </span></p><p class="c2"><span class="c0 c1">Colony pcr Master mix (batch of 70 amount in brackets)</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0">11.75 uL H2O (822.5 uL)-</span></p><p class="c2"><span class="c0">5 uL GOTAQ</span><span class="c0 c10"> 5X Reaction buffer </span><span class="c0">(350 uL)-</span></p><p class="c2"><span class="c0">2.5 uL Hyb-F forward primer (175 uL)-</span></p><p class="c2"><span class="c0">2.5 uL Ompa-R reverse primer (175 uL)-</span></p><p class="c2"><span class="c0">.5 uL dNTP 10 mM - (thawed & kept on ice) (35 uL)-</span></p><p class="c2"><span class="c0">0.25 uL polymerase enzyme, TAQ (17.5 uL)</span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0">2.5 uL of cells added to each respective PCR tube</span></p><p class="c2"><span class="c0 c10">Total Volume= 25 uL </span></p><p class="c2"><span class="c0 c10">Start: 3:31 pm</span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0 c10">10/20/2010</span></p><p class="c2"><span class="c0 c1">goals</span></p><ol class="c6"><li class="c8" value="1"><span class="c0">load rest of colony pcrs on gel from 10/19</span></li></ol><ol class="c3"><li class="c12" value="1"><span class="c0">load 5 ul</span></li></ol><p class="c2"><span class="c0 c1">protocols</span></p><p class="c2"><span class="c0 c1"> </span></p><p class="c2"><span class="c0 c1">loading gels for colony pcrs (Scott)</span></p><p class="c2"><span class="c0">expected fragments:</span></p><p class="c2"><span class="c0">hyb-f and ompa-r approx 500 bp. </span></p><p class="c2"><span class="c0">gel one (started 9:36 am)</span></p><p class="c2"><span class="c0 c4">order:</span></p><p class="c2"><span class="c0 c4">exact ladder|aoxa colony pcrs 1-6|ladder|aoxa colony pcrs 7-14|ladder</span></p><p class="c2"><img height="380.0" src="images/image2.png" width="508.0"></p><p class="c2"><span class="c0">gel two</span></p><p class="c2"><span class="c0 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="c2"><img height="387.0" src="images/image3.png" width="518.0"></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c4">gel three</span></p><p class="c2"><span class="c0 c4">order</span></p><p class="c2"><span class="c0 c4">exact ladder|aoxb colony pcrs 7-12 (from 10.19.2010)|ladder</span></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c4">gel four</span></p><p class="c2"><span class="c0 c4">order</span></p><p class="c2"><span class="c0 c4">exact ladder|aoxb colony pcrs 13-18 (from 10.19.2010)|ladder</span></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c4">(gel 3 and 4 are together below)</span></p><p class="c2"><img height="377.0" src="images/image6.png" width="504.0"></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c4">gel five</span></p><p class="c2"><span class="c0 c4">order</span></p><p class="c2"><span class="c0 c4">exact ladder|aoxb colony pcrs 19-20 (from 10.19.2010)|rfp-f3r colonies 1-4ladder</span></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c4">gel six</span></p><p class="c2"><span class="c0 c4">order</span></p><p class="c2"><span class="c0 c4">exact ladder|rfp-f3r colony pcrs 5-10(from 10.19.2010)|ladder</span></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c4">(gel 5 and 6 are below)</span></p><p class="c2"><img height="342.0" src="images/image4.png" width="458.0"></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c4">gel seven</span></p><p class="c2"><span class="c0 c4">order</span></p><p class="c2"><span class="c0 c4">exact ladder|rfp-f3r colony pcrs 10-20(from 10.19.2010)|ladder</span></p><p class="c2"><img height="311.0" src="images/image1.png" width="416.0"></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0 c1">RE Double Digest Recipe for pSB1C3 miniprep (from 10/19/2010) -- digest with speI and EcorI (Debika)</span></p><p class="c2"><span class="c0 c1">STARTED AT 2.54 PM</span></p><p class="c2"><span class="c0">12.5 uL H20 - </span></p><p class="c2"><span class="c0">3uL EcoRI Buffer -</span></p><p class="c2"><span class="c0">10 uL pSB1C3 (10.8.2010, 56 ng/uL)</span></p><p class="c2"><span class="c0">3 uL BSA (1ug/uL, to a final conc of .1mg/ml) -</span></p><p class="c2"><span class="c0">0.75 uL SpeI</span></p><p class="c2"><span class="c0">0.75 uL EcoRI</span></p><p class="c2"><span class="c0 c10">Total=30 ul total</span></p><p class="c2"><span class="c0">Digest overnight - put in waterbath 37 degrees at 2.54 PM</span></p><p class="c2"><span class="c0">Predict a insert (1000 bp) an(?- this was already there from the last protocol that Scott copied for me, DM)</span></p><p class="c2"><span class="c0 c4"> </span></p><p class="c2"><span class="c0 c10">Things to do</span></p><p class="c2"><span class="c0 c10">1. load colonies rfp-f3r 10-20 on gel, visualize for 500 bp band (done Christina)</span></p><p class="c2"><span class="c0 c10">2. colony pcr using aoxa/b f-R from colonies on 10/19/2010 (Done Christina)</span></p><p class="c2"><span class="c0 c10">3. grow up good colonies in 5ml liq culture</span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0 c1">colony pcr using aoxa/b f-R from colonies on 10/19/2010 </span></p><p class="c2"><span class="c0 c4">Do 2nd pcr on colonies (from 10/19/2010)</span></p><p class="c2"><span class="c0 c4">aoxa: 3,5,6</span></p><p class="c2"><span class="c0 c4">aoxb: 15,18</span></p><p class="c2"><span class="c0 c4">rfp-f3: 1, 11, 12, 18, 19</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0">Note: change the colony pcr program to have an extension time of 1.5 mins. </span></p><p class="c2"><span class="c0 c10">For aoxa (colonies 3, 5,6)</span></p><p class="c2"><span class="c0">11.75 uL H2O </span></p><p class="c2"><span class="c0">5 uL GOTAQ</span><span class="c0 c10"> 5X Reaction buffer </span></p><p class="c2"><span class="c0">2.5 uL Hyb F forward primer </span></p><p class="c2"><span class="c0">2.5 uL Aoxa R reverse primer </span></p><p class="c2"><span class="c0">.5 uL dNTP 10 mM - (thawed & kept on ice) </span></p><p class="c2"><span class="c0">0.25 uL polymerase enzyme, TAQ </span></p><p class="c2"><span class="c0">.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube</span></p><p class="c2"><span class="c0 c10">Total Volume= 25 uL </span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0 c10">For aoxb (colonies 15, 18)</span></p><p class="c2"><span class="c0">11.75 uL H2O </span></p><p class="c2"><span class="c0">5 uL GOTAQ</span><span class="c0 c10"> 5X Reaction buffer </span></p><p class="c2"><span class="c0">2.5 uL Hyb F forward primer </span></p><p class="c2"><span class="c0">2.5 uL Aoxb R reverse primer </span></p><p class="c2"><span class="c0">.5 uL dNTP 10 mM - (thawed & kept on ice) </span></p><p class="c2"><span class="c0">0.25 uL polymerase enzyme, TAQ </span></p><p class="c2"><span class="c0">.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube</span></p><p class="c2"><span class="c0 c10">Total Volume= 25 uL </span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0 c10">For RFP f3r (colonies 1, 11, 12, 18, 19)</span></p><p class="c2"><span class="c0">11.75 uL H2O </span></p><p class="c2"><span class="c0">5 uL GOTAQ</span><span class="c0 c10"> 5X Reaction buffer </span></p><p class="c2"><span class="c0">2.5 uL HybF forward primer </span></p><p class="c2"><span class="c0">2.5 uL Rfp-r reverse primer </span></p><p class="c2"><span class="c0">.5 uL dNTP 10 mM - (thawed & kept on ice) </span></p><p class="c2"><span class="c0">0.25 uL polymerase enzyme, TAQ </span></p><p class="c2"><span class="c0">.25 uL of pcr reaction from 10-20-10 added to each respective PCR tube</span></p><p class="c2"><span class="c0 c10">Total Volume= 25 uL </span></p><p class="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0 c10">*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="c2"><span class="c0 c10"> </span></p><p class="c2"><span class="c0 c1">Ligation of RFP-FR,HybB-FR,to psb1a3 (Christina)</span></p><p class="c2"><span class="c0 c4">Calculating equivalents: 9C</span></p><p class="c2"><span class="c0 c4">rfp (from9.22.2010)</span></p><p class="c2"><span class="c0 c4">psb1a3 (10.15, top )</span></p><p class="c2"><span class="c0 c4">hyb (from 9.10, purified 10.20)</span></p><p class="c2"><span class="c0">RFP- [41ng/uL]/678 bp= 0.06eq/uL</span></p><p class="c2"><span class="c0">hyBb-[ 6 ng/uL]/393 bp = 0.0152 eq/uL</span></p><p class="c2"><span class="c0">psb1a3-[ 9.4 ng/ul]/2000 bp= 0.0047 eq/uL</span></p><p class="c2"><span class="c0">for linear ligations, use a 2:2:1 of insert:insert:vector </span></p><p class="c2"><span class="c0 c1"> </span></p><p class="c2"><span class="c0">0.5 uL of RFP FR </span></p><p class="c2"><span class="c0">2 uL of HybB FR </span></p><p class="c2"><span class="c0">3 ul psb1a3</span></p><p class="c2"><span class="c0">1 uL 10x Ligase Buffer</span></p><p class="c2"><span class="c0">3 uL H20</span></p><p class="c2"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c2"><span class="c0 c10">Total=10 uL</span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0">overnight 4c </span></p><p class="c2"><span class="c0"> </span></p><p class="c2"><span class="c0">for tomorrow</span></p><ol class="c6"><li class="c8" value="1"><span class="c0">look on list for any overlaps and missed items</span></li><li class="c8"><span class="c0">note: make glycerol stocks when doing minipreps</span></li></ol> | ||
Revision as of 03:26, 21 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
- 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)
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)
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
