Team:GeorgiaTech/WeekThirteen

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<title>10/24-10/27</title><style type="text/css">ol{margin:0;padding:0}p{margin:0}.c2{vertical-align:top;width:93.6pt;border-style:solid;border-color:#000000;border-width:1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt}.c3{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c0{color:#ffffff;font-size:11pt;font-family:Arial}.c1{line-height:1.15;text-indent:0pt;direction:ltr}.c8{background-color:#ffffff}.c6{font-weight:bold}.c9{border-collapse:collapse}.c10{list-style-type:disc}.c4{text-decoration:underline}.c7{text-align:center}.c5{font-style:italic}</style></head><body class="c8"><p class="c1"><span class="c0 c6">10/24/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Plated RFP expression studies begun:</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="285.0" src="images/image0.png" width="342.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="275.0" src="images/image9.png" width="345.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="281.0" src="images/image8.png" width="343.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="279.0" src="images/image12.png" width="348.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="293.0" src="images/image2.png" width="339.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="299.0" src="images/image7.png" width="335.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="280.0" src="images/image13.png" width="355.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="262.0" src="images/image15.png" width="349.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="294.0" src="images/image1.png" width="344.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6">10/25/2010</span></p><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0 c6 c4">Goals</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">image hyb.ompa.rfp-f3r colony R11 plates to check for periplasmic expression (COMPLETED!)</span></li><li class="c3"><span class="c0">obtain SpeI from Hammer Lab (COMPLETED)</span></li><li class="c3"><span class="c0">digest A5 miniprep (10.23, in yellow rack) with EcoRI and SpeI (COMPLETED)</span></li><li class="c3"><span class="c0">make more gels</span></li><li class="c3"><span class="c0">this afternoon, gel extract the 1.5 kb isnert from A5 on a gel</span></li><li class="c3"><span class="c0">upload pictures from periplasmic expression to iGEM NB - send out an e-mail to all team members if possible to show these so that we can use the images for slides and poster.</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6 c4">Protocols</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Digest of colony A5 of hyb.ompa.aoxA.psb1a3 (from 10.23.2010)(Christina)</span></p><p class="c1"><span class="c0 c6">colony 5, Aox1a colony PCR from 10-22 (117 ng/ul)</span></p><p class="c1"><span class="c0">4.5 &nbsp;uL H20 -</span></p><p class="c1"><span class="c0">2uL 10X EcoRI buffer -</span></p><p class="c1"><span class="c0">10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.22.2010) -</span></p><p class="c1"><span class="c0">2 uL BSA (1ug/uL, to a final conc of .1mg/ml) &nbsp;-</span></p><p class="c1"><span class="c0">0.75 uL SpeI-</span></p><p class="c1"><span class="c0">0.75 uL EcoRI-</span></p><p class="c1"><span class="c0 c6">Total=20 ul total </span></p><p class="c1"><span class="c0">put on heat block at: 10:40 am</span></p><p class="c1"><span class="c0 c4"> </span></p><p class="c1"><span class="c0 c4">Imaging of the hyb.ompa.rfp-f3r R11 constructs</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><table cellpadding="0" cellspacing="0" class="c9"><tbody><tr><td class="c2"><p class="c1"><span class="c0">Condition</span></p></td><td class="c2"><p class="c1"><span class="c0">Filename</span></p></td><td class="c2"><p class="c1"><span class="c0">Observations</span></p></td><td class="c2"><p class="c1"><span class="c0">Slide Preparation</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Condition 8 (RT, 23c)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_1</span></p></td><td class="c2"><p class="c1"><span class="c0">Periplasmic expression</span></p></td><td class="c2"><p class="c1"><span class="c0">PBS, L-Lysine slide</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_2</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_3</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_4</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Condition 7 (RT, transferred to 37c)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_5</span></p></td><td class="c2"><p class="c1"><span class="c0">periplasmic expression but dimmer than 8</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Control (from 10.16 plate)(biobrick vector)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_6</span></p></td><td class="c2"><p class="c1"><span class="c0">cells are red throughout, appearing as solid ellipse </span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_7</span></p></td><td class="c2"><p class="c1"><span class="c0">cells are red throughout, appearing as solid ellipse </span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Cond 5 (20c, transferred to 37c)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_8</span></p></td><td class="c2"><p class="c1"><span class="c0">periplasmic expression</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">Same gain as iGEM-9</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Cond 6 (20c)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_9</span></p></td><td class="c2"><p class="c1"><span class="c0">periplasmic expression</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">same gain as iGEM-8</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_10</span></p></td><td class="c2"><p class="c1"><span class="c0">periplasmic expression</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">increase gain</span></p></td></tr></tbody></table><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Gel for gel extraction</span></p><p class="c1"><span class="c0 c5">order: gene exact ladder|digested psb1c3|digested A5 (10.23)|ladder|empty|digested psb1c3|digested A5 (from 10.23)</span></p><p class="c1"><span class="c0">started 5:20 pm</span></p><p class="c1"><img height="319.0" src="images/image10.png" width="426.0"></p><p class="c1"><span class="c0">(before gel extraction)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><img height="310.0" src="images/image14.png" width="415.0"></p><p class="c1"><span class="c0">After gel extraction</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Notes: the predicted 1.5 kb band was excised from the colony A5 hyb.ompa.aoxa digest, but the digest of the vector did not yield the predicted 2 kb backbone. </span></p><p class="c1"><span class="c0">Need to report the digest of psb1c3 with EcoRI and SpeI</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">hyb.ompa.aoxa A5 SpeI and EcoRI digest (2 kb band) gel extraction (Scott,Debika)</span></p><p class="c1"><span class="c0">1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.</span></p><p class="c1"><span class="c0">2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 &mu;L).</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Weight= 190 mg</span></p><p class="c1"><span class="c0">3. Incubated at 50&ordm;C for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 &ndash; 3 min during the incubation.</span></p><p class="c1"><span class="c0">4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).</span></p><p class="c1"><span class="c0">5. Added 1 gel volume of isopropanol to the sample and mixed.</span></p><p class="c1"><span class="c0">6. Placed a QIAquick spin column in a provided 2 mL collection tube.</span></p><p class="c1"><span class="c0">7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min @13,000k.</span></p><p class="c1"><span class="c0">8. Discarded flow-through and placed QIAquick column back in the same collection tube.</span></p><p class="c1"><span class="c0">9 To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min @13,000.</span></p><p class="c1"><span class="c0">10. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).</span></p><p class="c1"><span class="c0">11. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">12. To elute DNA, added 30 &nbsp;&mu;L water (pH 7.0 &ndash; 8.5), let the column stand for 1 min, and then centrifuged for 1 min @13,000k.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">RE Double Digest Recipe for pSB1C3 miniprep (from 10/19/2010) -- digest with speI and EcorI (Christina, Scott)</span></p><p class="c1"><span class="c0">12.5 uL H20 -</span></p><p class="c1"><span class="c0">3uL EcoRI Buffer - </span></p><p class="c1"><span class="c0">10 uL pSB1C3 (10.19.2010, 106 ng/uL)-</span></p><p class="c1"><span class="c0">3 uL BSA (1ug/uL, to a final conc of .1mg/ml) &nbsp;-</span></p><p class="c1"><span class="c0">0.75 uL SpeI-</span></p><p class="c1"><span class="c0">0.75 uL EcoRI-</span></p><p class="c1"><span class="c0 c6">Total=30 ul total</span></p><p class="c1"><span class="c0">Digest overnight - put in waterbath 37 degrees at 7:12 pm</span></p><p class="c1"><span class="c0">Predict a insert (1000 bp) and backbone (2kb)</span></p><p class="c1"><span class="c0">Nanospec: 27 ng/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">gel for gel extraction:</span></p><p class="c1"><span class="c0">ladder (exactgene)|4uL psb1c3|10 uL digested psb1c3 (10.25.2010)|10 uL digested psb1c3 (10.25.2010)</span></p><p class="c1"><img height="309.0" src="images/image11.png" width="441.0"></p><p class="c1"><span class="c0">(before gel extract)</span></p><p class="c1"><img height="309.0" src="images/image4.png" width="444.0"></p><p class="c1"><span class="c0">(after gel extract)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">psb1c3 (miniprep from 10.19.2010) SpeI and EcoRI digest (2 kb band) gel extraction (Repetition of the digest) (Christina, Scott)</span></p><p class="c1"><span class="c0">1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.</span></p><p class="c1"><span class="c0">2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 &mu;L).</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Weight= 290 mg</span></p><p class="c1"><span class="c0">3. Incubated at 50&ordm;C for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 &ndash; 3 min during the incubation.</span></p><p class="c1"><span class="c0">4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).</span></p><p class="c1"><span class="c0">5. Added 1 gel volume of isopropanol to the sample and mixed.</span></p><p class="c1"><span class="c0">6. Placed a QIAquick spin column in a provided 2 mL collection tube.</span></p><p class="c1"><span class="c0">7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min @13,000k.</span></p><p class="c1"><span class="c0">8. Discarded flow-through and placed QIAquick column back in the same collection tube.</span></p><p class="c1"><span class="c0">9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.</span></p><p class="c1"><span class="c0">10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min @13,000.</span></p><p class="c1"><span class="c0">11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).</span></p><p class="c1"><span class="c0">12. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">13. To elute DNA, added 30 &nbsp;&mu;L water (pH 7.0 &ndash; 8.5), let the column stand for 1 min, and then centrifuged for 1 min @13,000k.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Ligation of psb1c3 gel extraction (2 kb backbone) to gel extraction of hyb.ompa.aoxa colony A5 insert (1.5kb insert)</span></p><p class="c1"><span class="c0">use 2:1 product:vector</span></p><p class="c1"><span class="c0">Ligation:</span></p><p class="c1"><span class="c0">2 uL of insert (gel extraction from colony A5) &nbsp;1.5 kb = 0.0054 equivalents/uL -</span></p><p class="c1"><span class="c0">1.7 uL of vector (2kb backbone from gel extraction of psb1c3) 2 kb &nbsp;7.1 ng/ul = 0.0035 equivalents/uL-</span></p><p class="c1"><span class="c0">1 uL 10x T4 Ligase Buffer -</span></p><p class="c1"><span class="c0">4.8 uL H20 -</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c6">Total=10 uL</span></p><p class="c1"><span class="c0">leave at RT for 2 hours</span></p><p class="c1"><span class="c0">start 11:43 pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Started: 11:15am 10/26/10</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Heat shock transformation of the psb1c3+hyb.ompa.aoxa</span></p><p class="c1"><span class="c0">10 &#1405;L BL21 cells + 5 &#1405;L of ligation reaction</span></p><p class="c1"><span class="c0">(note, psb1c3 is choloamphenicol resistant!)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6 c5">See Protocols Page for Heat Shock Transformation</span></p><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0 c6">10/26/2010</span></p><p class="c1"><span class="c0 c6"> </span></p><p class="c1"><span class="c0 c4">Ligation of psb1c3 gel extraction (2 kb backbone) to gel extraction of hyb.ompa.aoxa colony A5 insert (1.5kb insert) &nbsp;(Christian)</span></p><p class="c1"><span class="c0">use 2:1 product:vector</span></p><p class="c1"><span class="c0">Ligation:</span></p><p class="c1"><span class="c0">2 uL of insert (gel extraction from colony A5) &nbsp;1.5 kb = 0.0054 equivalents/uL -</span></p><p class="c1"><span class="c0">1.7 uL of vector (2kb backbone from gel extraction of psb1c3) 2 kb &nbsp;7.1 ng/ul = 0.0035 equivalents/uL-</span></p><p class="c1"><span class="c0">1 uL 10x T4 Ligase Buffer -</span></p><p class="c1"><span class="c0">4.8 uL H20 -</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c6">Total=10 uL</span></p><p class="c1"><span class="c0">leave at RT for 2 hours</span></p><p class="c1"><span class="c0"> </span></p><p class="c1"><span class="c0">Plated at 7:15, placed in 37C</span></p><p class="c1"><span class="c0"> </span></p><p class="c1"><span class="c0 c4">Heat shock transformation of the psb1c3+hyb.ompa.aoxa (Scott)</span></p><p class="c1"><span class="c0">10 &#1405;L BL21 cells + 5 &#1405;L of ligation reaction</span></p><p class="c1"><span class="c0">(note, psb1c3 is choloamphenicol resistant!)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6 c5">See Protocols Page for Heat Shock Transformation</span></p><p class="c1"><span class="c0"> </span></p><p class="c1"><span class="c0 c4">picking colonies from psb1c3+hyb.ompa.aoxa a5 plates from 10.25.2010(Christina)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6">10/27/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Note: running low on miniprep spin columns and P2.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Miniprep of liquid cultures of psb1c3+hyb.ompa.aoxa A5 from 10/26</span></p><p class="c1"><span class="c0 c6 c5">See Protocols Page for Plasmid DNA Purification with Mini Prep Kit</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Picking colonies from psb1c3+hyb.ompa.aoxa a5 plates from 10.26.2010 (Christina, Scott)</span></p><p class="c1"><span class="c0">Picked 25 colonies, started 9 am</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Nanospec of minipreps of psb1c3+hyb.ompa.aoxa A5 (done today)</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c6 c4">Digests</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Digest of psb1c3+hyb.ompa.aoxa A5 from 10/27/2010 (minipreps from today)</span></p><p class="c1"><span class="c0">we are digesting minipreps 1-7:</span></p><p class="c1"><span class="c0">4.5 &nbsp;uL H20 -</span></p><p class="c1"><span class="c0">2uL 10X EcoRI buffer -</span></p><p class="c1"><span class="c0">10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.27.2010) (1-7)-</span></p><p class="c1"><span class="c0">2 uL BSA (1ug/uL, to a final conc of .1mg/ml) &nbsp;-</span></p><p class="c1"><span class="c0">0.75 uL SpeI</span></p><p class="c1"><span class="c0">0.75 uL EcoRI</span></p><p class="c1"><span class="c0 c6">Total=20 ul total</span></p><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0">put on heat block at 37c for 3 hours (start 10 am)</span></p><p class="c1"><span class="c0 c4">Digest of psb1c3+hyb.ompa.aoxa A5 from 10/27/2010 ( minipreps from today)</span></p><p class="c1"><span class="c0">we are digesting minipreps 1-</span></p><p class="c1"><span class="c0">5.25 &nbsp;uL H20</span></p><p class="c1"><span class="c0">2uL 10X EcoRI buffer</span></p><p class="c1"><span class="c0">10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.27.2010) (1-7)</span></p><p class="c1"><span class="c0">2 uL BSA (1ug/uL, to a final conc of .1mg/ml) &nbsp;</span></p><p class="c1"><span class="c0">0.75 uL EcoRI</span></p><p class="c1"><span class="c0 c6">Total=20 ul total</span></p><p class="c1"><span class="c0 c6">started 10:27 am</span></p><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0 c6">master mix</span></p><p class="c1"><span class="c0">7.5*(5.25)=39.375 ul h20</span></p><p class="c1"><span class="c0">7.5*(2)=15 ul buffer</span></p><p class="c1"><span class="c0">7.5*(2)=15 ul bsa</span></p><p class="c1"><span class="c0">7.5*(0.75)= 5.635 ul ecori</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6 c4">gels</span></p><p class="c1"><span class="c0">wells 1-7=digest of psb1c3 minipreps 1-7 from morning of 10/27 using EcorI and SpeI</span></p><p class="c1"><span class="c0">well 8=exactgene</span></p><p class="c1"><span class="c0">wells 9-15=digest of psb1c3 minipreps 1-7 from morning of 10/27 using EcorI</span></p><p class="c1"><span class="c0">(three versions of gel are presented below)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><img height="353.0" src="images/image6.png" width="440.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><img height="316.0" src="images/image3.png" width="442.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><img height="287.0" src="images/image5.png" width="444.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">pcr using RFP-FR and Hyb-f+aoxa-R (Debika, Scott)</span></p><p class="c1"><span class="c0">rxns</span></p><p class="c1"><span class="c0 c5">samples1-7 of psb1c3+hyb.ompa.aoxa</span></p><p class="c1"><span class="c0 c5">rxns:</span></p><p class="c1"><span class="c0 c5">1. rfp-FR (1-7 R)</span></p><p class="c1"><span class="c0 c5">2. Hyb-f Aoxa-R (8-14)</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">26.5 uL H2O-</span></li><li class="c3"><span class="c0">10 uL </span><span class="c0 c6">Taq 5X Reaction buffer-</span></li><li class="c3"><span class="c0">5 uL forward primer-</span></li><li class="c3"><span class="c0">5 uL reverse primer-</span></li><li class="c3"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)-</span></li><li class="c3"><span class="c0 c6">2 uL</span><span class="c0"> template DNA-</span></li><li class="c3"><span class="c0">0.5 uL polymerase enzyme, </span><span class="c0 c6">Gotaq</span></li></ol><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0 c6">Total Volume= 50 uL</span></p><p class="c1"><span class="c0 c6">1. master mix</span></p><p class="c1"><span class="c0">7.5*(26.5)=198.75 ul h20-</span></p><p class="c1"><span class="c0">7.5*(10)= 75 ul buffer-</span></p><p class="c1"><span class="c0">7.5*(5)= &nbsp;37.5 ul RFP-F-</span></p><p class="c1"><span class="c0">7.5*(5)= 37.5 ul RFP-R-</span></p><p class="c1"><span class="c0">7.5*(1)= 7.5ul dntp-</span></p><p class="c1"><span class="c0">7.5*(0.5)= 3.75ul taq-</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6">2. master mix</span></p><p class="c1"><span class="c0">7.5*(26.5)=198.75 ul h20-</span></p><p class="c1"><span class="c0">7.5*(10)= 75 ul buffer-</span></p><p class="c1"><span class="c0">7.5*(5)= &nbsp;37.5 ul Hyb-F-</span></p><p class="c1"><span class="c0">7.5*(5)= 37.5 ul aoxa-R-</span></p><p class="c1"><span class="c0">7.5*(1)= 7.5ul dntp-</span></p><p class="c1"><span class="c0">7.5*(0.5)= 3.75ul taq</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Colony PCR of transformations 5-10</span></p><p class="c1"><span class="c0">2 reactions:</span></p><p class="c1"><span class="c0 c5">1. rfp-FR (1-7 R)</span></p><p class="c1"><span class="c0 c5">2. Hyb-f omp-R (8-14)</span></p><p class="c1"><span class="c0">5 uL of cells</span></p><p class="c1"><span class="c0">25.5 uL H2O</span></p><p class="c1"><span class="c0">10 uL GOTAQ</span><span class="c0 c6"> 5X Reaction buffer</span></p><p class="c1"><span class="c0">5 uL forward primer</span></p><p class="c1"><span class="c0">5 uL &nbsp;reverse primer</span></p><p class="c1"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c1"><span class="c0">0.5 uL polymerase enzyme, TAQ</span></p><p class="c1"><span class="c0 c6">Total Volume= 50 uL</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Chose miniprep 3 (from this morning)(44ng/ul) to send off)</span></p><p class="c1"><span class="c0 c4">part=BBa_K410000.</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0">Tracking Number 796388355603</span></p>
+
<title>10/24-10/27</title><style type="text/css">ol{margin:0;padding:0}p{margin:0}.c2{vertical-align:top;width:93.6pt;border-style:solid;border-color:#000000;border-width:1.0pt;padding:5.0pt 5.0pt 5.0pt 5.0pt}.c3{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c0{color:#ffffff;font-size:11pt;font-family:Arial}.c1{line-height:1.15;text-indent:0pt;direction:ltr}.c8{background-color:#ffffff}.c6{font-weight:bold}.c9{border-collapse:collapse}.c10{list-style-type:disc}.c4{text-decoration:underline}.c7{text-align:center}.c5{font-style:italic}</style></head><body class="c8"><p class="c1"><span class="c0 c6">10/24/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Plated RFP expression studies begun:</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="285.0" src="http://2010.igem.org/wiki/images/5/51/10-24.0.png" width="342.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="275.0" src="images/image9.png" width="345.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="281.0" src="images/image8.png" width="343.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="279.0" src="images/image12.png" width="348.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="293.0" src="images/image2.png" width="339.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="299.0" src="images/image7.png" width="335.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="280.0" src="images/image13.png" width="355.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="262.0" src="images/image15.png" width="349.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1 c7"><img height="294.0" src="images/image1.png" width="344.0"></p><p class="c1 c7"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6">10/25/2010</span></p><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0 c6 c4">Goals</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">image hyb.ompa.rfp-f3r colony R11 plates to check for periplasmic expression (COMPLETED!)</span></li><li class="c3"><span class="c0">obtain SpeI from Hammer Lab (COMPLETED)</span></li><li class="c3"><span class="c0">digest A5 miniprep (10.23, in yellow rack) with EcoRI and SpeI (COMPLETED)</span></li><li class="c3"><span class="c0">make more gels</span></li><li class="c3"><span class="c0">this afternoon, gel extract the 1.5 kb isnert from A5 on a gel</span></li><li class="c3"><span class="c0">upload pictures from periplasmic expression to iGEM NB - send out an e-mail to all team members if possible to show these so that we can use the images for slides and poster.</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6 c4">Protocols</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Digest of colony A5 of hyb.ompa.aoxA.psb1a3 (from 10.23.2010)(Christina)</span></p><p class="c1"><span class="c0 c6">colony 5, Aox1a colony PCR from 10-22 (117 ng/ul)</span></p><p class="c1"><span class="c0">4.5 &nbsp;uL H20 -</span></p><p class="c1"><span class="c0">2uL 10X EcoRI buffer -</span></p><p class="c1"><span class="c0">10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.22.2010) -</span></p><p class="c1"><span class="c0">2 uL BSA (1ug/uL, to a final conc of .1mg/ml) &nbsp;-</span></p><p class="c1"><span class="c0">0.75 uL SpeI-</span></p><p class="c1"><span class="c0">0.75 uL EcoRI-</span></p><p class="c1"><span class="c0 c6">Total=20 ul total </span></p><p class="c1"><span class="c0">put on heat block at: 10:40 am</span></p><p class="c1"><span class="c0 c4"> </span></p><p class="c1"><span class="c0 c4">Imaging of the hyb.ompa.rfp-f3r R11 constructs</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><table cellpadding="0" cellspacing="0" class="c9"><tbody><tr><td class="c2"><p class="c1"><span class="c0">Condition</span></p></td><td class="c2"><p class="c1"><span class="c0">Filename</span></p></td><td class="c2"><p class="c1"><span class="c0">Observations</span></p></td><td class="c2"><p class="c1"><span class="c0">Slide Preparation</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Condition 8 (RT, 23c)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_1</span></p></td><td class="c2"><p class="c1"><span class="c0">Periplasmic expression</span></p></td><td class="c2"><p class="c1"><span class="c0">PBS, L-Lysine slide</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_2</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_3</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_4</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Condition 7 (RT, transferred to 37c)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_5</span></p></td><td class="c2"><p class="c1"><span class="c0">periplasmic expression but dimmer than 8</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Control (from 10.16 plate)(biobrick vector)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_6</span></p></td><td class="c2"><p class="c1"><span class="c0">cells are red throughout, appearing as solid ellipse </span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_7</span></p></td><td class="c2"><p class="c1"><span class="c0">cells are red throughout, appearing as solid ellipse </span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Cond 5 (20c, transferred to 37c)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_8</span></p></td><td class="c2"><p class="c1"><span class="c0">periplasmic expression</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">Same gain as iGEM-9</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">Cond 6 (20c)</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_9</span></p></td><td class="c2"><p class="c1"><span class="c0">periplasmic expression</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">same gain as iGEM-8</span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0">&nbsp;</span></p></td><td class="c2"><p class="c1"><span class="c0">iGEM_10</span></p></td><td class="c2"><p class="c1"><span class="c0">periplasmic expression</span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0">increase gain</span></p></td></tr></tbody></table><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Gel for gel extraction</span></p><p class="c1"><span class="c0 c5">order: gene exact ladder|digested psb1c3|digested A5 (10.23)|ladder|empty|digested psb1c3|digested A5 (from 10.23)</span></p><p class="c1"><span class="c0">started 5:20 pm</span></p><p class="c1"><img height="319.0" src="images/image10.png" width="426.0"></p><p class="c1"><span class="c0">(before gel extraction)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><img height="310.0" src="images/image14.png" width="415.0"></p><p class="c1"><span class="c0">After gel extraction</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Notes: the predicted 1.5 kb band was excised from the colony A5 hyb.ompa.aoxa digest, but the digest of the vector did not yield the predicted 2 kb backbone. </span></p><p class="c1"><span class="c0">Need to report the digest of psb1c3 with EcoRI and SpeI</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">hyb.ompa.aoxa A5 SpeI and EcoRI digest (2 kb band) gel extraction (Scott,Debika)</span></p><p class="c1"><span class="c0">1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.</span></p><p class="c1"><span class="c0">2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 &mu;L).</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Weight= 190 mg</span></p><p class="c1"><span class="c0">3. Incubated at 50&ordm;C for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 &ndash; 3 min during the incubation.</span></p><p class="c1"><span class="c0">4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).</span></p><p class="c1"><span class="c0">5. Added 1 gel volume of isopropanol to the sample and mixed.</span></p><p class="c1"><span class="c0">6. Placed a QIAquick spin column in a provided 2 mL collection tube.</span></p><p class="c1"><span class="c0">7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min @13,000k.</span></p><p class="c1"><span class="c0">8. Discarded flow-through and placed QIAquick column back in the same collection tube.</span></p><p class="c1"><span class="c0">9 To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min @13,000.</span></p><p class="c1"><span class="c0">10. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).</span></p><p class="c1"><span class="c0">11. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">12. To elute DNA, added 30 &nbsp;&mu;L water (pH 7.0 &ndash; 8.5), let the column stand for 1 min, and then centrifuged for 1 min @13,000k.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">RE Double Digest Recipe for pSB1C3 miniprep (from 10/19/2010) -- digest with speI and EcorI (Christina, Scott)</span></p><p class="c1"><span class="c0">12.5 uL H20 -</span></p><p class="c1"><span class="c0">3uL EcoRI Buffer - </span></p><p class="c1"><span class="c0">10 uL pSB1C3 (10.19.2010, 106 ng/uL)-</span></p><p class="c1"><span class="c0">3 uL BSA (1ug/uL, to a final conc of .1mg/ml) &nbsp;-</span></p><p class="c1"><span class="c0">0.75 uL SpeI-</span></p><p class="c1"><span class="c0">0.75 uL EcoRI-</span></p><p class="c1"><span class="c0 c6">Total=30 ul total</span></p><p class="c1"><span class="c0">Digest overnight - put in waterbath 37 degrees at 7:12 pm</span></p><p class="c1"><span class="c0">Predict a insert (1000 bp) and backbone (2kb)</span></p><p class="c1"><span class="c0">Nanospec: 27 ng/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">gel for gel extraction:</span></p><p class="c1"><span class="c0">ladder (exactgene)|4uL psb1c3|10 uL digested psb1c3 (10.25.2010)|10 uL digested psb1c3 (10.25.2010)</span></p><p class="c1"><img height="309.0" src="images/image11.png" width="441.0"></p><p class="c1"><span class="c0">(before gel extract)</span></p><p class="c1"><img height="309.0" src="images/image4.png" width="444.0"></p><p class="c1"><span class="c0">(after gel extract)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">psb1c3 (miniprep from 10.19.2010) SpeI and EcoRI digest (2 kb band) gel extraction (Repetition of the digest) (Christina, Scott)</span></p><p class="c1"><span class="c0">1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.</span></p><p class="c1"><span class="c0">2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 &mu;L).</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Weight= 290 mg</span></p><p class="c1"><span class="c0">3. Incubated at 50&ordm;C for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 &ndash; 3 min during the incubation.</span></p><p class="c1"><span class="c0">4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).</span></p><p class="c1"><span class="c0">5. Added 1 gel volume of isopropanol to the sample and mixed.</span></p><p class="c1"><span class="c0">6. Placed a QIAquick spin column in a provided 2 mL collection tube.</span></p><p class="c1"><span class="c0">7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min @13,000k.</span></p><p class="c1"><span class="c0">8. Discarded flow-through and placed QIAquick column back in the same collection tube.</span></p><p class="c1"><span class="c0">9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.</span></p><p class="c1"><span class="c0">10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min @13,000.</span></p><p class="c1"><span class="c0">11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).</span></p><p class="c1"><span class="c0">12. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">13. To elute DNA, added 30 &nbsp;&mu;L water (pH 7.0 &ndash; 8.5), let the column stand for 1 min, and then centrifuged for 1 min @13,000k.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Ligation of psb1c3 gel extraction (2 kb backbone) to gel extraction of hyb.ompa.aoxa colony A5 insert (1.5kb insert)</span></p><p class="c1"><span class="c0">use 2:1 product:vector</span></p><p class="c1"><span class="c0">Ligation:</span></p><p class="c1"><span class="c0">2 uL of insert (gel extraction from colony A5) &nbsp;1.5 kb = 0.0054 equivalents/uL -</span></p><p class="c1"><span class="c0">1.7 uL of vector (2kb backbone from gel extraction of psb1c3) 2 kb &nbsp;7.1 ng/ul = 0.0035 equivalents/uL-</span></p><p class="c1"><span class="c0">1 uL 10x T4 Ligase Buffer -</span></p><p class="c1"><span class="c0">4.8 uL H20 -</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c6">Total=10 uL</span></p><p class="c1"><span class="c0">leave at RT for 2 hours</span></p><p class="c1"><span class="c0">start 11:43 pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Started: 11:15am 10/26/10</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Heat shock transformation of the psb1c3+hyb.ompa.aoxa</span></p><p class="c1"><span class="c0">10 &#1405;L BL21 cells + 5 &#1405;L of ligation reaction</span></p><p class="c1"><span class="c0">(note, psb1c3 is choloamphenicol resistant!)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6 c5">See Protocols Page for Heat Shock Transformation</span></p><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0 c6">10/26/2010</span></p><p class="c1"><span class="c0 c6"> </span></p><p class="c1"><span class="c0 c4">Ligation of psb1c3 gel extraction (2 kb backbone) to gel extraction of hyb.ompa.aoxa colony A5 insert (1.5kb insert) &nbsp;(Christian)</span></p><p class="c1"><span class="c0">use 2:1 product:vector</span></p><p class="c1"><span class="c0">Ligation:</span></p><p class="c1"><span class="c0">2 uL of insert (gel extraction from colony A5) &nbsp;1.5 kb = 0.0054 equivalents/uL -</span></p><p class="c1"><span class="c0">1.7 uL of vector (2kb backbone from gel extraction of psb1c3) 2 kb &nbsp;7.1 ng/ul = 0.0035 equivalents/uL-</span></p><p class="c1"><span class="c0">1 uL 10x T4 Ligase Buffer -</span></p><p class="c1"><span class="c0">4.8 uL H20 -</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c6">Total=10 uL</span></p><p class="c1"><span class="c0">leave at RT for 2 hours</span></p><p class="c1"><span class="c0"> </span></p><p class="c1"><span class="c0">Plated at 7:15, placed in 37C</span></p><p class="c1"><span class="c0"> </span></p><p class="c1"><span class="c0 c4">Heat shock transformation of the psb1c3+hyb.ompa.aoxa (Scott)</span></p><p class="c1"><span class="c0">10 &#1405;L BL21 cells + 5 &#1405;L of ligation reaction</span></p><p class="c1"><span class="c0">(note, psb1c3 is choloamphenicol resistant!)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6 c5">See Protocols Page for Heat Shock Transformation</span></p><p class="c1"><span class="c0"> </span></p><p class="c1"><span class="c0 c4">picking colonies from psb1c3+hyb.ompa.aoxa a5 plates from 10.25.2010(Christina)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6">10/27/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Note: running low on miniprep spin columns and P2.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Miniprep of liquid cultures of psb1c3+hyb.ompa.aoxa A5 from 10/26</span></p><p class="c1"><span class="c0 c6 c5">See Protocols Page for Plasmid DNA Purification with Mini Prep Kit</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Picking colonies from psb1c3+hyb.ompa.aoxa a5 plates from 10.26.2010 (Christina, Scott)</span></p><p class="c1"><span class="c0">Picked 25 colonies, started 9 am</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Nanospec of minipreps of psb1c3+hyb.ompa.aoxa A5 (done today)</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c6 c4">Digests</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Digest of psb1c3+hyb.ompa.aoxa A5 from 10/27/2010 (minipreps from today)</span></p><p class="c1"><span class="c0">we are digesting minipreps 1-7:</span></p><p class="c1"><span class="c0">4.5 &nbsp;uL H20 -</span></p><p class="c1"><span class="c0">2uL 10X EcoRI buffer -</span></p><p class="c1"><span class="c0">10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.27.2010) (1-7)-</span></p><p class="c1"><span class="c0">2 uL BSA (1ug/uL, to a final conc of .1mg/ml) &nbsp;-</span></p><p class="c1"><span class="c0">0.75 uL SpeI</span></p><p class="c1"><span class="c0">0.75 uL EcoRI</span></p><p class="c1"><span class="c0 c6">Total=20 ul total</span></p><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0">put on heat block at 37c for 3 hours (start 10 am)</span></p><p class="c1"><span class="c0 c4">Digest of psb1c3+hyb.ompa.aoxa A5 from 10/27/2010 ( minipreps from today)</span></p><p class="c1"><span class="c0">we are digesting minipreps 1-</span></p><p class="c1"><span class="c0">5.25 &nbsp;uL H20</span></p><p class="c1"><span class="c0">2uL 10X EcoRI buffer</span></p><p class="c1"><span class="c0">10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.27.2010) (1-7)</span></p><p class="c1"><span class="c0">2 uL BSA (1ug/uL, to a final conc of .1mg/ml) &nbsp;</span></p><p class="c1"><span class="c0">0.75 uL EcoRI</span></p><p class="c1"><span class="c0 c6">Total=20 ul total</span></p><p class="c1"><span class="c0 c6">started 10:27 am</span></p><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0 c6">master mix</span></p><p class="c1"><span class="c0">7.5*(5.25)=39.375 ul h20</span></p><p class="c1"><span class="c0">7.5*(2)=15 ul buffer</span></p><p class="c1"><span class="c0">7.5*(2)=15 ul bsa</span></p><p class="c1"><span class="c0">7.5*(0.75)= 5.635 ul ecori</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6 c4">gels</span></p><p class="c1"><span class="c0">wells 1-7=digest of psb1c3 minipreps 1-7 from morning of 10/27 using EcorI and SpeI</span></p><p class="c1"><span class="c0">well 8=exactgene</span></p><p class="c1"><span class="c0">wells 9-15=digest of psb1c3 minipreps 1-7 from morning of 10/27 using EcorI</span></p><p class="c1"><span class="c0">(three versions of gel are presented below)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><img height="353.0" src="images/image6.png" width="440.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><img height="316.0" src="images/image3.png" width="442.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><img height="287.0" src="images/image5.png" width="444.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">pcr using RFP-FR and Hyb-f+aoxa-R (Debika, Scott)</span></p><p class="c1"><span class="c0">rxns</span></p><p class="c1"><span class="c0 c5">samples1-7 of psb1c3+hyb.ompa.aoxa</span></p><p class="c1"><span class="c0 c5">rxns:</span></p><p class="c1"><span class="c0 c5">1. rfp-FR (1-7 R)</span></p><p class="c1"><span class="c0 c5">2. Hyb-f Aoxa-R (8-14)</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">26.5 uL H2O-</span></li><li class="c3"><span class="c0">10 uL </span><span class="c0 c6">Taq 5X Reaction buffer-</span></li><li class="c3"><span class="c0">5 uL forward primer-</span></li><li class="c3"><span class="c0">5 uL reverse primer-</span></li><li class="c3"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)-</span></li><li class="c3"><span class="c0 c6">2 uL</span><span class="c0"> template DNA-</span></li><li class="c3"><span class="c0">0.5 uL polymerase enzyme, </span><span class="c0 c6">Gotaq</span></li></ol><p class="c1"><span class="c0 c6">&nbsp;</span></p><p class="c1"><span class="c0 c6">Total Volume= 50 uL</span></p><p class="c1"><span class="c0 c6">1. master mix</span></p><p class="c1"><span class="c0">7.5*(26.5)=198.75 ul h20-</span></p><p class="c1"><span class="c0">7.5*(10)= 75 ul buffer-</span></p><p class="c1"><span class="c0">7.5*(5)= &nbsp;37.5 ul RFP-F-</span></p><p class="c1"><span class="c0">7.5*(5)= 37.5 ul RFP-R-</span></p><p class="c1"><span class="c0">7.5*(1)= 7.5ul dntp-</span></p><p class="c1"><span class="c0">7.5*(0.5)= 3.75ul taq-</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c6">2. master mix</span></p><p class="c1"><span class="c0">7.5*(26.5)=198.75 ul h20-</span></p><p class="c1"><span class="c0">7.5*(10)= 75 ul buffer-</span></p><p class="c1"><span class="c0">7.5*(5)= &nbsp;37.5 ul Hyb-F-</span></p><p class="c1"><span class="c0">7.5*(5)= 37.5 ul aoxa-R-</span></p><p class="c1"><span class="c0">7.5*(1)= 7.5ul dntp-</span></p><p class="c1"><span class="c0">7.5*(0.5)= 3.75ul taq</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">Colony PCR of transformations 5-10</span></p><p class="c1"><span class="c0">2 reactions:</span></p><p class="c1"><span class="c0 c5">1. rfp-FR (1-7 R)</span></p><p class="c1"><span class="c0 c5">2. Hyb-f omp-R (8-14)</span></p><p class="c1"><span class="c0">5 uL of cells</span></p><p class="c1"><span class="c0">25.5 uL H2O</span></p><p class="c1"><span class="c0">10 uL GOTAQ</span><span class="c0 c6"> 5X Reaction buffer</span></p><p class="c1"><span class="c0">5 uL forward primer</span></p><p class="c1"><span class="c0">5 uL &nbsp;reverse primer</span></p><p class="c1"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c1"><span class="c0">0.5 uL polymerase enzyme, TAQ</span></p><p class="c1"><span class="c0 c6">Total Volume= 50 uL</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Chose miniprep 3 (from this morning)(44ng/ul) to send off)</span></p><p class="c1"><span class="c0 c4">part=BBa_K410000.</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0">Tracking Number 796388355603</span></p>

Revision as of 00:34, 28 October 2010

Georgia Institute of Technology iGEM Team 2010 Homepage

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10/24-10/27

10/24/2010

 

Plated RFP expression studies begun:

 

 

 

 

 

 

 

 

 

 

10/25/2010

 

Goals

  1. image hyb.ompa.rfp-f3r colony R11 plates to check for periplasmic expression (COMPLETED!)
  2. obtain SpeI from Hammer Lab (COMPLETED)
  3. digest A5 miniprep (10.23, in yellow rack) with EcoRI and SpeI (COMPLETED)
  4. make more gels
  5. this afternoon, gel extract the 1.5 kb isnert from A5 on a gel
  6. upload pictures from periplasmic expression to iGEM NB - send out an e-mail to all team members if possible to show these so that we can use the images for slides and poster.

 

 

Protocols

 

Digest of colony A5 of hyb.ompa.aoxA.psb1a3 (from 10.23.2010)(Christina)

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

put on heat block at: 10:40 am

Imaging of the hyb.ompa.rfp-f3r R11 constructs

 

 

Condition

Filename

Observations

Slide Preparation

 

Condition 8 (RT, 23c)

iGEM_1

Periplasmic expression

PBS, L-Lysine slide

 

 

iGEM_2

 

-

 

 

iGEM_3

 

-

 

 

iGEM_4

 

-

 

Condition 7 (RT, transferred to 37c)

iGEM_5

periplasmic expression but dimmer than 8

-

 

Control (from 10.16 plate)(biobrick vector)

iGEM_6

cells are red throughout, appearing as solid ellipse

-

 

 

iGEM_7

cells are red throughout, appearing as solid ellipse

-

 

Cond 5 (20c, transferred to 37c)

iGEM_8

periplasmic expression

-

Same gain as iGEM-9

Cond 6 (20c)

iGEM_9

periplasmic expression

-

same gain as iGEM-8

 

iGEM_10

periplasmic expression

-

increase gain

 

Gel for gel extraction

order: gene exact ladder|digested psb1c3|digested A5 (10.23)|ladder|empty|digested psb1c3|digested A5 (from 10.23)

started 5:20 pm

(before gel extraction)

 

After gel extraction

 

Notes: the predicted 1.5 kb band was excised from the colony A5 hyb.ompa.aoxa digest, but the digest of the vector did not yield the predicted 2 kb backbone.

Need to report the digest of psb1c3 with EcoRI and SpeI

 

hyb.ompa.aoxa A5 SpeI and EcoRI digest (2 kb band) gel extraction (Scott,Debika)

1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).

        Weight= 190 mg

3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.

4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).

5. Added 1 gel volume of isopropanol to the sample and mixed.

6. Placed a QIAquick spin column in a provided 2 mL collection tube.

7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min @13,000k.

8. Discarded flow-through and placed QIAquick column back in the same collection tube.

9 To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min @13,000.

10. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).

11. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.

12. To elute DNA, added 30  μL water (pH 7.0 – 8.5), let the column stand for 1 min, and then centrifuged for 1 min @13,000k.

 

RE Double Digest Recipe for pSB1C3 miniprep (from 10/19/2010) -- digest with speI and EcorI (Christina, Scott)

12.5 uL H20 -

3uL EcoRI Buffer -

10 uL pSB1C3 (10.19.2010, 106 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 7:12 pm

Predict a insert (1000 bp) and backbone (2kb)

Nanospec: 27 ng/uL

 

gel for gel extraction:

ladder (exactgene)|4uL psb1c3|10 uL digested psb1c3 (10.25.2010)|10 uL digested psb1c3 (10.25.2010)

(before gel extract)

(after gel extract)

 

psb1c3 (miniprep from 10.19.2010) SpeI and EcoRI digest (2 kb band) gel extraction (Repetition of the digest) (Christina, Scott)

1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.

2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μL).

        Weight= 290 mg

3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.

4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).

5. Added 1 gel volume of isopropanol to the sample and mixed.

6. Placed a QIAquick spin column in a provided 2 mL collection tube.

7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min @13,000k.

8. Discarded flow-through and placed QIAquick column back in the same collection tube.

9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.

10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min @13,000.

11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).

12. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.

13. To elute DNA, added 30  μL water (pH 7.0 – 8.5), let the column stand for 1 min, and then centrifuged for 1 min @13,000k.

 

Ligation of psb1c3 gel extraction (2 kb backbone) to gel extraction of hyb.ompa.aoxa colony A5 insert (1.5kb insert)

use 2:1 product:vector

Ligation:

2 uL of insert (gel extraction from colony A5)  1.5 kb = 0.0054 equivalents/uL -

1.7 uL of vector (2kb backbone from gel extraction of psb1c3) 2 kb  7.1 ng/ul = 0.0035 equivalents/uL-

1 uL 10x T4 Ligase Buffer -

4.8 uL H20 -

0.5 uL T4 Ligase

Total=10 uL

leave at RT for 2 hours

start 11:43 pm

 

Started: 11:15am 10/26/10

 

Heat shock transformation of the psb1c3+hyb.ompa.aoxa

10 սL BL21 cells + 5 սL of ligation reaction

(note, psb1c3 is choloamphenicol resistant!)

 

See Protocols Page for Heat Shock Transformation

 

10/26/2010

Ligation of psb1c3 gel extraction (2 kb backbone) to gel extraction of hyb.ompa.aoxa colony A5 insert (1.5kb insert)  (Christian)

use 2:1 product:vector

Ligation:

2 uL of insert (gel extraction from colony A5)  1.5 kb = 0.0054 equivalents/uL -

1.7 uL of vector (2kb backbone from gel extraction of psb1c3) 2 kb  7.1 ng/ul = 0.0035 equivalents/uL-

1 uL 10x T4 Ligase Buffer -

4.8 uL H20 -

0.5 uL T4 Ligase

Total=10 uL

leave at RT for 2 hours

Plated at 7:15, placed in 37C

Heat shock transformation of the psb1c3+hyb.ompa.aoxa (Scott)

10 սL BL21 cells + 5 սL of ligation reaction

(note, psb1c3 is choloamphenicol resistant!)

 

See Protocols Page for Heat Shock Transformation

picking colonies from psb1c3+hyb.ompa.aoxa a5 plates from 10.25.2010(Christina)

 

10/27/2010

 

Note: running low on miniprep spin columns and P2.

 

Miniprep of liquid cultures of psb1c3+hyb.ompa.aoxa A5 from 10/26

See Protocols Page for Plasmid DNA Purification with Mini Prep Kit

 

Picking colonies from psb1c3+hyb.ompa.aoxa a5 plates from 10.26.2010 (Christina, Scott)

Picked 25 colonies, started 9 am

 

Nanospec of minipreps of psb1c3+hyb.ompa.aoxa A5 (done today)

 

Digests

 

Digest of psb1c3+hyb.ompa.aoxa A5 from 10/27/2010 (minipreps from today)

we are digesting minipreps 1-7:

4.5  uL H20 -

2uL 10X EcoRI buffer -

10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.27.2010) (1-7)-

2 uL BSA (1ug/uL, to a final conc of .1mg/ml)  -

0.75 uL SpeI

0.75 uL EcoRI

Total=20 ul total

 

put on heat block at 37c for 3 hours (start 10 am)

Digest of psb1c3+hyb.ompa.aoxa A5 from 10/27/2010 ( minipreps from today)

we are digesting minipreps 1-

5.25  uL H20

2uL 10X EcoRI buffer

10 uL hyb.ompa.aoxa.psb1a3 colony 5 miniprep (10.27.2010) (1-7)

2 uL BSA (1ug/uL, to a final conc of .1mg/ml)  

0.75 uL EcoRI

Total=20 ul total

started 10:27 am

 

master mix

7.5*(5.25)=39.375 ul h20

7.5*(2)=15 ul buffer

7.5*(2)=15 ul bsa

7.5*(0.75)= 5.635 ul ecori

 

gels

wells 1-7=digest of psb1c3 minipreps 1-7 from morning of 10/27 using EcorI and SpeI

well 8=exactgene

wells 9-15=digest of psb1c3 minipreps 1-7 from morning of 10/27 using EcorI

(three versions of gel are presented below)

 

 

 

 

pcr using RFP-FR and Hyb-f+aoxa-R (Debika, Scott)

rxns

samples1-7 of psb1c3+hyb.ompa.aoxa

rxns:

1. rfp-FR (1-7 R)

2. Hyb-f Aoxa-R (8-14)

  1. 26.5 uL H2O-
  2. 10 uL Taq 5X Reaction buffer-
  3. 5 uL forward primer-
  4. 5 uL reverse primer-
  5. 1 uL dNTP 10 mM - (thawed & kept on ice)-
  6. 2 uL template DNA-
  7. 0.5 uL polymerase enzyme, Gotaq

 

Total Volume= 50 uL

1. master mix

7.5*(26.5)=198.75 ul h20-

7.5*(10)= 75 ul buffer-

7.5*(5)=  37.5 ul RFP-F-

7.5*(5)= 37.5 ul RFP-R-

7.5*(1)= 7.5ul dntp-

7.5*(0.5)= 3.75ul taq-

 

2. master mix

7.5*(26.5)=198.75 ul h20-

7.5*(10)= 75 ul buffer-

7.5*(5)=  37.5 ul Hyb-F-

7.5*(5)= 37.5 ul aoxa-R-

7.5*(1)= 7.5ul dntp-

7.5*(0.5)= 3.75ul taq

 

Colony PCR of transformations 5-10

2 reactions:

1. rfp-FR (1-7 R)

2. Hyb-f omp-R (8-14)

5 uL of cells

25.5 uL H2O

10 uL GOTAQ 5X Reaction buffer

5 uL forward primer

5 uL  reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

0.5 uL polymerase enzyme, TAQ

Total Volume= 50 uL

 

Chose miniprep 3 (from this morning)(44ng/ul) to send off)

part=BBa_K410000.

 

Tracking Number 796388355603