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2010-10-28T02:58:59Z
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Aschwartz9 at 01:10, 28 October 2010
2010-10-28T01:10:32Z
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Aschwartz9 at 00:41, 28 October 2010
2010-10-28T00:41:07Z
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2010-10-28T00:34:36Z
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Aschwartz9: New page: <html xmlns="http://www.w3.org/1999/xhtml"> <head> <meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /> <title>Georgia Institute of Technology iGEM Team 2010 Homepage</tit...
2010-10-28T00:24:49Z
<p>New page: <html xmlns="http://www.w3.org/1999/xhtml"> <head> <meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /> <title>Georgia Institute of Technology iGEM Team 2010 Homepage</tit...</p>
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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></div>
Aschwartz9