Team:Grinnell/Notebook/Gels

From 2011.igem.org

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<h1>Gel Pics</h1>
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<h1>Gel Pics</h1>
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<div style="float:right; position:relative; left:-125px; top:-5px; z-index:-1">
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<h2>Week 1</h2>
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<img alt="Our first gel" src='https://static.igem.org/mediawiki/2011/3/32/20110606_PCRproduct.jpg' height=200px />
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<p>We collected more gel data than we could fit on one page. Please go on to one of the following pages:</p>
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<a name="20110606_PCRproduct" href="https://2011.igem.org/File:20110606_PCRproduct.jpg">
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    <li><a href="https://2011.igem.org/Team:Grinnell/Notebook/Gels/DspB">DspB</a></li>
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<img alt="20110606_PCRproduct" src="https://static.igem.org/mediawiki/2011/3/32/20110606_PCRproduct.jpg" />
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    <li><a href="https://2011.igem.org/Team:Grinnell/Notebook/Gels/Esp">Esp</a></li>
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    <li><a href="https://2011.igem.org/Team:Grinnell/Notebook/Gels/Promoters">Promoters</a></li>
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<a name="20110610PlamidGel" href="https://2011.igem.org/File:20110610PlamidGel.jpg">
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PCR products on DNA gel. Lane 1: ladder; Lane 2: <i>rsaA</i> from liquid culture <i>Caulobacter</i>; Lane 3: <i>rsaA</i> from plate culture <i>Caulobacter</i>; Lane 4: <i>esp</i> from <i>S. epidermidis</i>.
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Gel results for transformation of ligations. Lane 1: ladder; Lane 2: digested plasmid with <i>rsaA</i> C-terminal; Lane 3: digested plasmid with <i>rsaA</i> C-terminal and <i>esp</i>; Lanes 4-8: digested plasmids from various colonies with <i>esp</i>.
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<h2>Week 2</h2>
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<a name="20110614_PCR135" href="https://2011.igem.org/File:20110614_PCR135.jpg">
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<a name="20110614_PCR246" href="https://2011.igem.org/File:20110614_PCR246.jpg">
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<img alt="20110614_PCR246" src="https://static.igem.org/mediawiki/2011/1/1d/20110614_PCR246.jpg">
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<a name="20110614_Promoters" href="https://2011.igem.org/File:20110614_Promoters.jpg">
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<img alt="20110614_Promoters" src="https://static.igem.org/mediawiki/2011/7/76/20110614_Promoters.jpg">
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<a name="20110616_promotorGel" href="https://2011.igem.org/File:20110616_promotorGel.jpg">
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<img alt="20110616_promotorGel" src="https://static.igem.org/mediawiki/2011/9/9e/20110616_promotorGel.jpg">
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Gel 1 of 2. Lane 1: ladder; lanes 2-4: PCR product using VF2 and VR of plasmid containing <i>esp</i> ligated with <i>rsaA</i> C-terminal that had been digested 20110606; lane 5: PCR product of plasmid containing <i>esp</i>; lanes 6-8: PCR product using VF2 and VR of plasmid containing <i>esp</i> ligated with <i>rsaA</i> C-terminal PCR product from 20110605. Only lane 6 shows successful ligation of <i>esp</i> and <i>rsaA</i> C-terminal.
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Gel 2 of 2.  Lane 1: ladder; lanes 2-4: PCR product using VF2 and VR of plasmid containing <i>esp</i> ligated with <i>rsaA</i> C-terminal that had been digested 20110606; lane 5: PCR product of plasmid containing <i>esp</i>; lanes 6-8: PCR product using VF2 and VR of plasmid containing <i>esp</i> ligated with <i>rsaA</i> C-terminal PCR product from 20110605.  None of these show successful ligation.
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Gel of PCR of <i>Caulobacter</i> promoters Pxyl (inducible) and PrsaA (constitutive). Lane 1: ladder; lane 2: PrsaA; Lane 3: Pxyl. Bands appear, but are hazy and spread out due to the small size of DNA fragments.
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PCR products of <i>Caulobacter</i> promoters PrsaA and Pxyl.  Lane 1: ladder; lane 2: PrsaA; lane 3: Pxyl.
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<a name="20110616_promoterPostCleanup" href="https://2011.igem.org/File:20110616_promoterPostCleanup.jpg">
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<img alt="20110616_promoterPostCleanup" src="https://static.igem.org/mediawiki/2011/2/2e/20110616_promoterPostCleanup.jpg">
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<a name="20110617_Pro-esp-stop-rsaA" href="https://2011.igem.org/File:20110617_Pro-esp-stop-rsaA.jpg">
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<img alt="20110617_Pro-esp-stop-rsaA" src="https://static.igem.org/mediawiki/2011/d/d6/20110617_Pro-esp-stop-rsaA.jpg">
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<a name="20110617_rsaA%26esp" href="https://2011.igem.org/File:20110617_rsaA%26esp.jpg">
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<img alt="20110617_rsaA%26esp" src="https://static.igem.org/mediawiki/2011/d/dd/20110617_rsaA%26esp.jpg">
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Gel confirming that clean up did not remove DNA fragments. Lane 1: ladder; lane 2: PrsaA; lane 3: Pxyl.
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Gel of PCR products of transformations of insertion of Biobrick promoter BBa_K081005 into pSB1C3 containing esp and rsaA C-terminal. Lane 1: ladder; lanes 2-4 and 6-8: PCR product of transformation survivors; lane 5: control DNA of <i>esp</i> + <i>rsaA</i> C-terminal. Only lane 2 shows something that may be success, but as the fragment for the promoter is so small, it is difficult to confirm based solely on gel electrophoresis.
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Gel of PCR product of <i>esp</i> and <i>rsaA</i> C-terminal using new primers. Lane 1: ladder; lanes 2,3: PCR product of <i>rsaA</i> from chromosomal <i>Caulobacter</i> DNA; lane 4: PCR product of <i>esp</i> using new primers from chromosomal <i>S. epidermidis</i> DNA.
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<h2>Week3</h2>
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<a name='20110620_promotorsTransformationGel' href='https://2011.igem.org/File:20110620_promotorsTransformationGel.jpg'>
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<img alt='20110620_promotorsTransformationGel' src='https://static.igem.org/mediawiki/2011/4/4c/20110620_promotorsTransformationGel.jpg' />
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<a name='20110621_BBaGel1' href='https://2011.igem.org/File:20110621_BBaGel1.jpg'>
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<img alt='20110621 BBaGel1' src='https://static.igem.org/mediawiki/2011/4/43/20110621_BBaGel1.jpg' />
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<a name='20110621_espAndCombo' href='https://2011.igem.org/File:20110621_espAndCombo.jpg'>
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<img alt='20110621 espAndCombo' src='https://static.igem.org/mediawiki/2011/b/bf/20110621_espAndCombo.jpg' />
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<a name='20110621_Promoter-esp-Stop-rsaA-pMR10' href='https://2011.igem.org/File:20110621_Promoter-esp-Stop-rsaA-pMR10.jpg'>
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<img alt='20110621 Promoter-esp-Stop-rsaA-pMR10' src='https://static.igem.org/mediawiki/2011/3/3b/20110621_Promoter-esp-Stop-rsaA-pMR10.jpg' />
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Gel of Plasmid PCR of promoters. Presence of two small bands shows contamination of samples. Lane 1: ladder; lanes 2-5: plasmid PCR of transformation survivors.
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Gel of BBa_K081005 digests from minipreps. Lane 1: ladder; lane 2: digest of miniprep from overnights that should carry the desired promoter insert. Digest shows one band: the linearized plasmid.
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Lanes 1-6: plasmid PCR of transformed cells that should carry the desired <i>esp</i> insert; lane 7: ladder; lanes 8-10: plasmid PCR of transformed cells that should be carrying <i>esp</i> + <i>rsaA</i> C-terminal insert.  A few of the <i>esp</i> inserts succeeded, but as expected the three piece ligation failed again.
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Lanes 1-3 and 5-8: plasmid PCR of transformed cells that should carry the Promoter-esp-stop-rsaA insert in plasmid pMR10; lane 4: ladder.  Only lane 3 shows any success.
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<a name='20110621_rsaA' href='https://2011.igem.org/File:20110621_rsaA.jpg'>
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<img alt='20110621 rsaA' src='https://static.igem.org/mediawiki/2011/1/1f/20110621_rsaA.jpg' />
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<a name='20110622_PromotersGel2' href='https://2011.igem.org/File:20110622_PromotersGel2.jpg'>
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<img alt='20110622_PromotersGel2' src='https://static.igem.org/mediawiki/2011/a/a3/20110622_PromotersGel2.jpg' />
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<a name='20110623_esp%2BrsaA_135' href='https://2011.igem.org/File:20110623_esp%2BrsaA_135.jpg'>
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<img alt='20110623_esp%2BrsaA_135' src='https://static.igem.org/mediawiki/2011/a/a3/20110623_esp%2BrsaA_135.jpg' />
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<a name='20110623_esp%2BrsaA_246' href='https://2011.igem.org/File:20110623_esp%2BrsaA_246.jpg'>
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<img alt='20110623_esp%2BrsaA_246' src='https://static.igem.org/mediawiki/2011/a/aa/20110623_esp%2BrsaA_246.jpg' />
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Lanes 1-3 and 5-7: plasmid PCR of colonies that should be carrying ''rsaA'' C-terminal insert in pSB1C3; lane 4: ladder.  Lanes 3 and 4 seem to show insert at significant concentrations.
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</td>
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Lane 1: ladder; lanes 2-4: plasmid PCR product of various promoter insertions into pSB1C3. Smearing due to leftover circular plasmid. Insertions seem to have been successful.
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Gel 1 of 2. Colony PCR of transformation products of insertions of <i>esp</i> from PCR into pSB1C3 containing <i>rsaA</i> C-terminal, insertions of <i>rsaA</i> C-terminal into pSB1C3 containing <i>esp</i>, and <i>esp</i> and <i>rsaA</i> C-terminal digests from pSB1C3 into pMR10 as a three piece ligation. Lane 1: ladder; lanes 2-4: <i>esp</i> from PCR inserted into <i>rsaA</i> containing pSB1C3; lanes 5-7: <i>rsaA</i> from PCR inserted into <i>esp</i> containing pSB1C3; lanes 8-10: <i>esp</i> and <i>rsaA</i> digests from plasmid insertion into pMR10. Lanes 2-4 show correct insertion length, but none of the other fragments are the correct length.
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Gel 2 of 2. Colony PCR of transformation products of insertions of <i>esp</i> from PCR into pSB1C3 containing <i>rsaA</i> C-terminal, insertions of <i>rsaA</i> C-terminal into pSB1C3 containing <i>esp</i>, and <i>esp</i> and <i>rsaA</i> C-terminal digests from pSB1C3 into pMR10 as a three piece ligation. Lane 1: ladder; lanes 2-4: <i>esp</i> from PCR inserted into <i>rsaA</i> containing pSB1C3; lanes 5-7: <i>rsaA</i> from PCR inserted into <i>esp</i> containing pSB1C3; lanes 8-10: <i>esp</i> and <i>rsaA</i> digests from plasmid insertion into pMR10. Lanes 2-4 show correct insertion length, but none of the other fragments are the correct length.
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<a name='20110624_PrsaA_esp_rsaA' href='https://2011.igem.org/File:20110624_PrsaA_esp_rsaA.jpg'>
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<img alt='20110624_PrsaA_esp_rsaA' src='https://static.igem.org/mediawiki/2011/2/2a/20110624_PrsaA_esp_rsaA.jpg' />
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<a name='20110624_Pxyl_esp_rsaA' href='https://2011.igem.org/File:20110624_Pxyl_esp_rsaA.jpg'>
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<img alt='20110624_Pxyl_esp_rsaA' src='https://static.igem.org/mediawiki/2011/5/5e/20110624_Pxyl_esp_rsaA.jpg' />
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<a name='20110624_BBa_K081005_esp_rsaA' href='https://2011.igem.org/File:20110624_BBa_K081005_esp_rsaA.jpg'>
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<img alt='20110624_BBa_K081005_esp_rsaA' src='https://static.igem.org/mediawiki/2011/e/e9/20110624_BBa_K081005_esp_rsaA.jpg' />
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Colony PCR products of transformation products of insertions of <i>esp</i> + <i>rsaA</i> C-terminal into plasmid containing <i>Caulobacter</i> constitutive promoter PrsaA. Lane 1: ladder; lanes 2-7: PCR products; lane 8: standard PCR product of pSB1C3 containing only PrsaA.  Transformation was successful, but ligation, and perhaps digestion, were not.
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Colony PCR products of transformation products of insertion of <i>esp</i> + <i>rsaA</i> into pSB1C3 containing <i>Caulobacter</i> inducible promoter Pxyl (induced in presence of xylene).  Lane 1: ladder; lanes 2-7: PCR products; lane 8: standard PCR product of pSB1C3 containing only Pxyl.  Transformation was successful, but ligation was not.
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Colony PCR amplified DNA fragments of transformation products of inserts into plasmid containing existing Biobrick promoter BBa_K081005. Lane 1: ladder; lanes 2-7: PCR products; lane 8: standard DNA fragment identified as containing BBa_K081005. The lack of DNA may be due to a breakdown in our antibiotic; the transformation is being repeated.
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<h2>Week 4</h2>
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<table class='gallery' frame='void' rules='none'>
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<a name='20110627_esprsaA%2Bpromoters_1' href='https://2011.igem.org/File:20110627_esprsaA%2Bpromoters_1.jpg'>
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<img alt='20110627_esprsaA%2Bpromoters_1' src='https://static.igem.org/mediawiki/2011/3/3c/20110627_esprsaA%2Bpromoters_1.jpg' />
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<img alt='20110627_esprsaA%2Bpromoters_2' src='https://static.igem.org/mediawiki/2011/0/0f/20110627_esprsaA%2Bpromoters_2.jpg' />
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<a name='20110628_TestDigest' href='https://2011.igem.org/File:20110628_TestDigest.jpg'>
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<a name='20110629_BBa_K081005' href='https://2011.igem.org/File:20110629_BBa_K081005.jpg'>
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<img alt='20110629_BBa_K081005' src='https://static.igem.org/mediawiki/2011/3/31/20110629_BBa_K081005.jpg' />
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Gel 1 of 2.  PCR products of transformation cells that should contain <i>esp</i> + <i>rsaA</i> C-terminal insert into plasmids containing various promoters. Lane 1: ladder; lanes 2-5: inserts into plasmid containing BBa_K081005; lane 6: standard BBa_K081005 containing no insert; lanes 7-10: inserts into plasmid containing P<sub>xyl</sub>.  While the BBa_K081005 plasmids seem to have some insert, none of these show an insert of appropriate size (~1.3-1.5kb).
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Gel 2 of 2. PCR products of transformation cells that should contain <i>esp</i> + <i>rsaA</i> C-terminal insert into plasmids containing various promoters. Lane 1: ladder; lanes 2-7: inserts into plasmid containing P<sub>rsaA</sub>; lane 8: standard P<sub>rsaA</sub> with no insert; lane 9: standard P<sub>xyl</sub> with no insert. The first lane shows odd results, but none of these seems to have the desired insert.
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Test to ensure all of our restriction enzymes are still active.  Lane 1: ladder; lane 2: cut with Eco RI; lane 3: cut with XbaI; lane 4: standard uncut DNA fragment for lanes 2 and 3; lane 5: cut with SpeI; lane 6: cut with PstI; lane 7: standard uncut DNA fragment for lanes 5 and 6.  There is an apparent decrease in size from standard DNA fragments to the digested samples.  In the digest lanes there is also a dim band fairly far down the gel corresponding to the small end piece that is the other product of digestion.
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Lane 1: ladder; lane 2: colony PCR product for promoter BBa_K081005 in preparation for digestion and insertion into plasmid containing <i>esp</i> and <i>rsaA</i> C-terminal.  Result is positive.
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<a name='20110629_PromoterInserts1' href='https://2011.igem.org/File:20110629_PromoterInserts1.jpg'>
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<a name='20110630_espORcombo' href='https://2011.igem.org/File:20110630_espORcombo.jpg'>
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Gel 1 of 3. PCR of transformation cells that should contain an insert of P<sub>xyl</sub> into pSB1C3 containing <i>esp</i> and <i>rsaA</i> C-terminal. Lane 1: ladder; lanes 2-5: PCR with plasmid primers VF2 and VR; lanes 6-9: PCR with promoter specific forward primer and plasmid reverse primer VR.  The lack of DNA is disturbing and suggests that our plates are ineffective or contaminated.
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</td>
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<td>
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Gel 2 of 3. PCR of transformation cells that should contain an insert of P<sub>rsaA</sub> into pSB1C3 containing <i>esp</i> and <i>rsaA</i> C-terminal. Lane 1: ladder; lanes 2-5: PCR with plasmid primers VF2 and VR; lanes 6-9: PCR with promoter specific forward primer and plasmid reverse primer VR. Bands in lanes 2, 3, and 4 suggests that these colonies at least contained the plasmid, but the apparent size of the band is too small for <i>esp</i> and <i>rsaA</i> C-terminal together; rather it is the correct size for just <i>rsaA</i> C-terminal.
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</td>
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<td>
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Gel 3 of 3. PCR product of transformation colonies that should contain an insert of either P<sub>xyl</sub> or P<sub>rsaA</sub> into pSB1C3 containing <i>esp</i> and <i>rsaA</i> C-terminal. Lane 1: ladder; lanes 2,3: P<sub>xyl</sub> insert PCR using plasmid primers VF2 and VR; lanes 4,5: P<sub>rsaA</sub> insert PCR using plasmid primers VF2 and VR; lanes 6,7: P<sub>xyl</sub> insert PCR using promoter specific forward primer and VR; lanes 8,9: P<sub>rsaA</sub> insert PCR using promoter specific forward primer and VR. Lanes 8 and 9 suggest a successful insertion of P<sub>rsaA</sub>, however all of the bands are too small.
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</td>
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<td>
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Verification of presence of lack of <i>rsaA</i> C-terminal insert in pSB1C3 containing <i>esp</i>.  Lane 1: ladder; lane 2: digested PCR of possible insert; lane 3: standard digested <i>esp</i>. The band is faint, but there appears to be a band at the same height as the standard as well as a couple higher bands for leftover plasmids, suggesting that there was no insertion of <i>rsaA</i> C-terminal.
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</td>
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</tr>
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<td>
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<a name='20110701_combo1' href='https://2011.igem.org/File:20110701_combo1.jpg'>
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<img alt='20110701_combo1' src='https://static.igem.org/mediawiki/2011/b/b2/20110701_combo1.jpg' />
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</a>
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<td>
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<a name='20110701_combo2' href='https://2011.igem.org/File:20110701_combo2.jpg'>
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<img alt='20110701_combo2' src='https://static.igem.org/mediawiki/2011/4/42/20110701_combo2.jpg' />
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</a>
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Gel 1 of 2. PCR of transformation products of <i>rsaA</i> C-terminal insert into pSB1C3 containing <i>esp</i>. Lane 1: ladder; lanes 2-7: ligation 1, colonies 1-6; lanes 8-10: ligation 2, colonies 1-3.  The complete lack of DNA fragments here means that these ligations failed.
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</td>
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<td>
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Gel 2 of 2. PCR of transformation products of <i>rsaA</i> C-terminal insert into pSB1C3 containing <i>esp</i>. Lane 1: ladder; lanes 2-4: ligation 2, colonies 4-6; lanes 5-10: ligation 3, colonies 1-6.  All the bands here are correct for <i>esp</i>, but not the combination.
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</td>
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</tr>
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</table>
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<!--###############################-->
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<h2>Week 5</h2>
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<table class='gallery' frame='void' rules='none'>
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<a name='20110704_combo' href='https://2011.igem.org/File:20110704_combo.jpg'>
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<img alt='20110704_combo' src='https://static.igem.org/mediawiki/2011/f/f8/20110704_combo.jpg' />
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</a>
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<td>
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PCR test of transformation products of insertion of gel extracted DNA into plasmid containing another gene. Lane 1: ladder; lanes 2-4: insertion of <i>rsaA</i> C-terminal into plasmid containing <i>esp</i>; lanes 5-7: insertion of <i>esp</i> into plasmid containing <i>rsaA</i> C-terminal.  Smearing of bands is inconclusive and suggests that there may be no DNA (colonies are contaminants) or that freeze-thaw did not succeed in providing sufficient template DNA for PCR.
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</td>
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</tr>
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</table>
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</body></html>
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Latest revision as of 15:55, 25 July 2011

Grinnell Menubar

Gel Pics

Our first gel

We collected more gel data than we could fit on one page. Please go on to one of the following pages:

Retrieved from "http://2011.igem.org/Team:Grinnell/Notebook/Gels"