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| ==2011.07.03-2011.07.09== | | ==2011.07.03-2011.07.09== |
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| + | <html> |
| + | <table class="gallery" frame='void' rules='none'> |
| + | <tr> |
| + | <td> |
| + | <a name='20110704_combo' href='https://2011.igem.org/File:20110704_combo.jpg'> |
| + | <img alt='20110704_combo' src='https://static.igem.org/mediawiki/2011/f/f8/20110704_combo.jpg' /> |
| + | </a> |
| + | </td> |
| + | <td> |
| + | <a name='20110705_notCombo' href='https://2011.igem.org/File:20110705_notCombo.jpg'> |
| + | <img alt ='20110705_notCombo' src='https://static.igem.org/mediawiki/2011/5/55/20110705_notCombo.jpg' /> |
| + | </a> |
| + | </td> |
| + | <td> |
| + | <a name='20110705_Tests' href='https://2011.igem.org/File:20110705_Tests.jpg'> |
| + | <img alt ='20110705_Tests' src='https://static.igem.org/mediawiki/2011/e/e0/20110705_Tests.jpg' /> |
| + | </a> |
| + | </td> |
| + | <td> |
| + | <a name='20110705_TestGelExtract' href='https://2011.igem.org/File:20110705_TestGelExtract.jpg'> |
| + | <img alt ='20110705_TestGelExtract' src='https://static.igem.org/mediawiki/2011/2/2f/20110705_TestGelExtract.jpg' /> |
| + | </a> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td> |
| + | 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. |
| + | </td> |
| + | <td> |
| + | Digested miniprep samples of overnight cultures of transformation product that may contain <i>esp</i> and <i>rsaA</i>. Lane 1: <i>esp</i> standard; lanes 2,3: <i>rsaA</i> from gel extraction inserted into pSB1C3 containing <i>esp</i> from plates spread with 20μL of transformation cells; lanes 4,5: <i>rsaA</i> from gel extraction inserted into pSB1C3 containing <i>esp</i> from plates spread with 200μL of transformation cells; lanes 6,7: <i>esp</i> from gel extraction inserted into pSB1C3 containing <i>rsaA</i> from plates spread with 20μL of transformation cells; lanes 8,9: <i>esp</i> from gel extraction inserted into pSB1C3 containing <i>rsaA</i> from plates spread with 200μL of transformation cells; lane 10: ladder. |
| + | </td> |
| + | <td> |
| + | Lane 1: ladder; lane 2: digest of miniprep of transformation product of insert of <i>esp</i> from gel extraction into plasmid containing <i>rsaA</i>; lanes 3,4: ligations gel extracted <i>esp</i> and <i>rsaA</i> and plasmid containing the other gene; lane 5: ligation of gel extracted <i>esp</i> with <i>rsaA</i>. Lane 2 shows that ligation was unsuccessful. The rest of the lanes are inconclusive and a follow up gel with additional DNA is to be run. |
| + | </td> |
| + | <td> |
| + | Lane 1: ladder; lane 2: ligation of <i>esp</i> and <i>rsaA</i> C-terminal fragments from gel extraction. No <i>rsaA</i> appears to be present, nor any ligation, only <i>esp</i>. |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td> |
| + | <a name='20110706_espGelExtractPrep' href='https://2011.igem.org/File:20110706_espGelExtractPrep.jpg'> |
| + | <img alt ='20110706_espGelExtractPrep' src='https://static.igem.org/mediawiki/2011/d/dc/20110706_espGelExtractPrep.jpg' /> |
| + | </a> |
| + | </td> |
| + | <td> |
| + | <a name='20110706_rsaAGelExtractPrep' href='https://2011.igem.org/File:20110706_rsaAGelExtractPrep.jpg'> |
| + | <img alt ='20110706_rsaAGelExtractPrep' src='https://static.igem.org/mediawiki/2011/2/21/20110706_rsaAGelExtractPrep.jpg' /> |
| + | </a> |
| + | </td> |
| + | <td> |
| + | <a name='20110706_MoreColonies' href='https://2011.igem.org/File:20110706_MoreColonies.jpg'> |
| + | <img alt ='20110706_MoreColonies' src='https://static.igem.org/mediawiki/2011/4/4c/20110706_MoreColonies.jpg' /> |
| + | </a> |
| + | </td> |
| + | <td> |
| + | <a name='20110706_MoreColoniesFollowup' href='https://2011.igem.org/File:20110706_MoreColoniesFollowup.jpg'> |
| + | <img alt ='20110706_MoreColoniesFollowup' src='https://static.igem.org/mediawiki/2011/2/20/20110706_MoreColoniesFollowup.jpg' /> |
| + | </a> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td> |
| + | Gel of digested pSB1C3 containing <i>esp</i> for gel extraction. Lane 1: ladder; lanes 2,3: digest with EcoRI and SpeI; lanes 5,6: digest with SpeI and PstI. |
| + | </td> |
| + | <td> |
| + | Gel of digests of pSB1C3 containing <i>rsaA</i> C-terminal in preparation for gel extraction. Lane 1: ladder; lanes 3,4: digest with XbaI and PstI; lanes 6,7: digest with EcoRI and XbaI. |
| + | </td> |
| + | <td> |
| + | Gel of restriction digests (EcoRI and PstI) of miniprepped DNA from more transformants from the weekend's transformations. |
| + | Lane 1: ladder; lanes 2-5: colonies that should contain <i>rsaA</i> inserted into pSB1C3 containing <i>esp</i>; lanes 6-9: colonies that should contain <i>esp</i> inserted into pSB1C3 containing <i>rsaA</i>; lane 10: <i>esp</i> standard. |
| + | </td> |
| + | <td> |
| + | Follow up gel of lanes 5 and 6 from the previous gel, redigested for a longer period of time. Lane 1: ladder; lane 2: digest of miniprep product from colony that should contain an insert of <i>rsaA</i> into plasmid already containing <i>esp</i> (previous lane 5); lane 3: digest of miniprep product from colony that should contain an insert of <i>esp</i> into plasmid already containing <i>rsaA</i> (previous lane 6). |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td> |
| + | <a name='20110707_GXandPCR_combo' href='https://2011.igem.org/File:20110707_GXandPCR_combo.jpg'> |
| + | <img alt ='20110707_GXandPCR_combo' src='https://static.igem.org/mediawiki/2011/2/25/20110707_GXandPCR_combo.jpg' /> |
| + | </a> |
| + | </td> |
| + | <td> |
| + | <a name='20110708_BBaGel' href='https://2011.igem.org/File:20110708_BBaGel.jpg'> |
| + | <img alt ='20110708_BBaGel' src='https://static.igem.org/mediawiki/2011/a/a8/20110708_BBaGel.jpg' /> |
| + | </a> |
| + | </td> |
| + | <td> |
| + | <a name='20110708_Pxyl_PrsaA_combo' href='https://2011.igem.org/File:20110708_Pxyl_PrsaA_combo.jpg'> |
| + | <img alt ='20110708_Pxyl_PrsaA_combo' src='https://static.igem.org/mediawiki/2011/4/47/20110708_Pxyl_PrsaA_combo.jpg' /> |
| + | </a> |
| + | </td> |
| + | <td> |
| + | <a name='20110709_MorePromoter_combo_1' href='https://2011.igem.org/File:20110709_MorePromoter_combo_1.jpg'> |
| + | <img alt ='20110709_MorePromoter_combo_1' src='https://static.igem.org/mediawiki/2011/e/e6/20110709_MorePromoter_combo_1.jpg' /> |
| + | </a> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td> |
| + | Gel for gel extraction of <i>esp</i> and <i>rsaA</i> combination and checking the identity of PCR product. Lane 1: ladder; lanes 3,4: digest of miniprep DNA for gel extraction; lane 6: PCR product. Gel is somewhat messy, but appropriately sized bands are apparent. |
| + | </td> |
| + | <td> |
| + | Gel of transformation products of <i>esp</i> + <i>rsaA</i> insert into plasmid containing promoter BBa_K081005. Lane 1: ladder; lanes 2-5: transformants using gel extracted combo gene; lanes 6-9: transformants using PCR product for combo insert. |
| + | </td> |
| + | <td> |
| + | Gel of transformants that should carry <i>esp</i> + <i>rsaA</i> behind a promoter. Lane 1: ladder; lanes 2-5: P<sub>rsaA</sub> with insert; lanes 6-9: P<sub>xyl</sub> with insert. None of these transformants show the desired insert. |
| + | </td> |
| + | <td> |
| + | Gel of more transformants that should have <i>esp</i> + <i>rsaA</i> combo inserted behind P<sub>xyl</sub>. Lane 1: ladder; lanes 2-4: combo from miniprep digest and gel extract insert; lanes 5-7: combo from PCR insert; lane 8: confirmation of insertion behind BBa_K081005; lane 9: standard combo from gel extraction. |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td> |
| + | <a name='20110709_MorePromoter_combo_2' href='https://2011.igem.org/File:20110709_MorePromoter_combo_2.jpg'> |
| + | <img alt ='20110709_MorePromoter_combo_2' src='https://static.igem.org/mediawiki/2011/a/af/20110709_MorePromoter_combo_2.jpg' /> |
| + | </a> |
| + | </td> |
| + | </tr> |
| + | <tr> |
| + | <td> |
| + | Gel of transformants that should carry <i>esp</i> + <i>rsaA</i> combo insert behind P<sub>rsaA</sub> run against standard combo. Lane 1: ladder; lanes 2-4: combo from miniprep digest and gel extract insert; lanes 5-7: combo from PCR product insert; lane 8: standard combo without any promoter. |
| + | </td> |
| + | </tr> |
| + | </table> |
| + | </html> |
| + | |
| + | ==2011.07.10-2011.07.16== |
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Lanes 1-6: plasmid PCR of transformed cells that should carry the desired esp insert; lane 7: ladder; lanes 8-10: plasmid PCR of transformed cells that should be carrying esp + rsaA C-terminal insert. A few of the esp 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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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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Gel 1 of 2. Colony PCR of transformation products of insertions of esp from PCR into pSB1C3 containing rsaA C-terminal, insertions of rsaA C-terminal into pSB1C3 containing esp, and esp and rsaA C-terminal digests from pSB1C3 into pMR10 as a three piece ligation. Lane 1: ladder; lanes 2-4: esp from PCR inserted into rsaA containing pSB1C3; lanes 5-7: rsaA from PCR inserted into esp containing pSB1C3; lanes 8-10: esp and rsaA 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 esp from PCR into pSB1C3 containing rsaA C-terminal, insertions of rsaA C-terminal into pSB1C3 containing esp, and esp and rsaA C-terminal digests from pSB1C3 into pMR10 as a three piece ligation. Lane 1: ladder; lanes 2-4: esp from PCR inserted into rsaA containing pSB1C3; lanes 5-7: rsaA from PCR inserted into esp containing pSB1C3; lanes 8-10: esp and rsaA 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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Colony PCR products of transformation products of insertions of esp + rsaA C-terminal into plasmid containing Caulobacter 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 esp + rsaA into pSB1C3 containing Caulobacter 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 esp + rsaA 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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Gel 1 of 2. PCR products of transformation cells that should contain esp + rsaA 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 Pxyl. 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 esp + rsaA C-terminal insert into plasmids containing various promoters. Lane 1: ladder; lanes 2-7: inserts into plasmid containing PrsaA; lane 8: standard PrsaA with no insert; lane 9: standard Pxyl with no insert. The first lane shows odd results, but none of these seems to have the desired insert.
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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 esp and rsaA C-terminal. Result is positive.
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Gel 1 of 3. PCR of transformation cells that should contain an insert of Pxyl into pSB1C3 containing esp and rsaA 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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Gel 2 of 3. PCR of transformation cells that should contain an insert of PrsaA into pSB1C3 containing esp and rsaA 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 esp and rsaA C-terminal together; rather it is the correct size for just rsaA C-terminal.
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Gel 3 of 3. PCR product of transformation colonies that should contain an insert of either Pxyl or PrsaA into pSB1C3 containing esp and rsaA C-terminal. Lane 1: ladder; lanes 2,3: Pxyl insert PCR using plasmid primers VF2 and VR; lanes 4,5: PrsaA insert PCR using plasmid primers VF2 and VR; lanes 6,7: Pxyl insert PCR using promoter specific forward primer and VR; lanes 8,9: PrsaA insert PCR using promoter specific forward primer and VR. Lanes 8 and 9 suggest a successful insertion of PrsaA, however all of the bands are too small.
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Verification of presence of lack of rsaA C-terminal insert in pSB1C3 containing esp. Lane 1: ladder; lane 2: digested PCR of possible insert; lane 3: standard digested esp. 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 rsaA C-terminal.
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Gel 1 of 2. PCR of transformation products of rsaA C-terminal insert into pSB1C3 containing esp. 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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Gel 2 of 2. PCR of transformation products of rsaA C-terminal insert into pSB1C3 containing esp. 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 esp, but not the combination.
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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 rsaA C-terminal into plasmid containing esp; lanes 5-7: insertion of esp into plasmid containing rsaA 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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Digested miniprep samples of overnight cultures of transformation product that may contain esp and rsaA. Lane 1: esp standard; lanes 2,3: rsaA from gel extraction inserted into pSB1C3 containing esp from plates spread with 20μL of transformation cells; lanes 4,5: rsaA from gel extraction inserted into pSB1C3 containing esp from plates spread with 200μL of transformation cells; lanes 6,7: esp from gel extraction inserted into pSB1C3 containing rsaA from plates spread with 20μL of transformation cells; lanes 8,9: esp from gel extraction inserted into pSB1C3 containing rsaA from plates spread with 200μL of transformation cells; lane 10: ladder.
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Lane 1: ladder; lane 2: digest of miniprep of transformation product of insert of esp from gel extraction into plasmid containing rsaA; lanes 3,4: ligations gel extracted esp and rsaA and plasmid containing the other gene; lane 5: ligation of gel extracted esp with rsaA. Lane 2 shows that ligation was unsuccessful. The rest of the lanes are inconclusive and a follow up gel with additional DNA is to be run.
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Lane 1: ladder; lane 2: ligation of esp and rsaA C-terminal fragments from gel extraction. No rsaA appears to be present, nor any ligation, only esp.
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Gel of digested pSB1C3 containing esp for gel extraction. Lane 1: ladder; lanes 2,3: digest with EcoRI and SpeI; lanes 5,6: digest with SpeI and PstI.
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Gel of digests of pSB1C3 containing rsaA C-terminal in preparation for gel extraction. Lane 1: ladder; lanes 3,4: digest with XbaI and PstI; lanes 6,7: digest with EcoRI and XbaI.
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Gel of restriction digests (EcoRI and PstI) of miniprepped DNA from more transformants from the weekend's transformations.
Lane 1: ladder; lanes 2-5: colonies that should contain rsaA inserted into pSB1C3 containing esp; lanes 6-9: colonies that should contain esp inserted into pSB1C3 containing rsaA; lane 10: esp standard.
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Follow up gel of lanes 5 and 6 from the previous gel, redigested for a longer period of time. Lane 1: ladder; lane 2: digest of miniprep product from colony that should contain an insert of rsaA into plasmid already containing esp (previous lane 5); lane 3: digest of miniprep product from colony that should contain an insert of esp into plasmid already containing rsaA (previous lane 6).
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Gel for gel extraction of esp and rsaA combination and checking the identity of PCR product. Lane 1: ladder; lanes 3,4: digest of miniprep DNA for gel extraction; lane 6: PCR product. Gel is somewhat messy, but appropriately sized bands are apparent.
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Gel of transformation products of esp + rsaA insert into plasmid containing promoter BBa_K081005. Lane 1: ladder; lanes 2-5: transformants using gel extracted combo gene; lanes 6-9: transformants using PCR product for combo insert.
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Gel of transformants that should carry esp + rsaA behind a promoter. Lane 1: ladder; lanes 2-5: PrsaA with insert; lanes 6-9: Pxyl with insert. None of these transformants show the desired insert.
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Gel of more transformants that should have esp + rsaA combo inserted behind Pxyl. Lane 1: ladder; lanes 2-4: combo from miniprep digest and gel extract insert; lanes 5-7: combo from PCR insert; lane 8: confirmation of insertion behind BBa_K081005; lane 9: standard combo from gel extraction.
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Gel of transformants that should carry esp + rsaA combo insert behind PrsaA run against standard combo. Lane 1: ladder; lanes 2-4: combo from miniprep digest and gel extract insert; lanes 5-7: combo from PCR product insert; lane 8: standard combo without any promoter.
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