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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"> </span></p><p class="c1"><span class="c0">Plated RFP expression studies begun:</span></p><p class="c1"><span class="c0"> </span></p><p class="c1 c7"><img height="285.0" src="https://static.igem.org/mediawiki/2010/5/51/10-24.0.png" width="342.0"></p><p class="c1 c7"><span class="c0"> </span></p><p class="c1 c7"><img height="275.0" src="images/image9.png" width="345.0"></p><p class="c1"><span class="c0"> </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"> </span></p><p class="c1 c7"><img height="279.0" src="images/image12.png" width="348.0"></p><p class="c1"><span class="c0"> </span></p><p class="c1 c7"><img height="293.0" src="images/image2.png" width="339.0"></p><p class="c1"><span class="c0"> </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"> </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"> </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"> </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"> </span></p><p class="c1"><span class="c0 c6">10/25/2010</span></p><p class="c1"><span class="c0 c6"> </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"> </span></p><p class="c1"><span class="c0"> </span></p><p class="c1"><span class="c0 c6 c4">Protocols</span></p><p class="c1"><span class="c0 c4"> </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 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) -</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"> </span></p><p class="c1"><span class="c0"> </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"> </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"> </span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0"> </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"> </span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0"> </span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0"> </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"> </span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0"> </span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0"> </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"> </span></p></td><td class="c2"><p class="c1"><span class="c0">-</span></p></td><td class="c2"><p class="c1"><span class="c0"> </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"> </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"> </span></p></td></tr><tr><td class="c2"><p class="c1"><span class="c0"> </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"> </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"> </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"> </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"> </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"> </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"> </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 μL).</span></p><p class="c1"><span class="c0"> Weight= 190 mg</span></p><p class="c1"><span class="c0">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.</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 μL water (pH 7.0 – 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"> </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) -</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"> </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"> </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 μL).</span></p><p class="c1"><span class="c0"> Weight= 290 mg</span></p><p class="c1"><span class="c0">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.</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 μL water (pH 7.0 – 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"> </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) 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 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"> </span></p><p class="c1"><span class="c0">Started: 11:15am 10/26/10</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</span></p><p class="c1"><span class="c0">10 սL BL21 cells + 5 սL of ligation reaction</span></p><p class="c1"><span class="c0">(note, psb1c3 is choloamphenicol resistant!)</span></p><p class="c1"><span class="c0"> </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"> </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) (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) 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 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 սL BL21 cells + 5 սL of ligation reaction</span></p><p class="c1"><span class="c0">(note, psb1c3 is choloamphenicol resistant!)</span></p><p class="c1"><span class="c0"> </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"> </span></p><p class="c1"><span class="c0 c6">10/27/2010</span></p><p class="c1"><span class="c0"> </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"> </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"> </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"> </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"> </span></p><p class="c1"><span class="c0 c6 c4">Digests</span></p><p class="c1"><span class="c0 c4"> </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 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) -</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"> </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 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) </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"> </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"> </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"> </span></p><p class="c1"><img height="353.0" src="images/image6.png" width="440.0"></p><p class="c1"><span class="c0"> </span></p><p class="c1"><img height="316.0" src="images/image3.png" width="442.0"></p><p class="c1"><span class="c0"> </span></p><p class="c1"><img height="287.0" src="images/image5.png" width="444.0"></p><p class="c1"><span class="c0"> </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 & 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"> </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)= 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"> </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)= 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"> </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 reverse primer</span></p><p class="c1"><span class="c0">1 uL dNTP 10 mM - (thawed & 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"> </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"> </span></p><p class="c1"><span class="c0">Tracking Number 796388355603</span></p> | |
Revision as of 00:34, 28 October 2010
10/24/2010
Plated RFP expression studies begun:
10/25/2010
Goals
- image hyb.ompa.rfp-f3r colony R11 plates to check for periplasmic expression (COMPLETED!)
- obtain SpeI from Hammer Lab (COMPLETED)
- digest A5 miniprep (10.23, in yellow rack) with EcoRI and SpeI (COMPLETED)
- make more gels
- this afternoon, gel extract the 1.5 kb isnert from A5 on a gel
- 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)
- 26.5 uL H2O-
- 10 uL Taq 5X Reaction buffer-
- 5 uL forward primer-
- 5 uL reverse primer-
- 1 uL dNTP 10 mM - (thawed & kept on ice)-
- 2 uL template DNA-
- 0.5 uL polymerase enzyme, Gotaq
Total Volume= 50 uL
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
