Team:Grinnell CN/Notebook/Gels/Esp

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Latest revision as of 04:14, 27 September 2011

Gels for Esp

2011.06.05-2011.06.11

20110606_PCRproduct 20110610PlamidGel
PCR products on DNA gel. Lane 1: ladder; Lane 2: rsaA from liquid culture Caulobacter; Lane 3: rsaA from plate culture Caulobacter; Lane 4: esp from S. epidermidis. Gel results for transformation of ligations. Lane 1: ladder; Lane 2: digested plasmid with rsaA C-terminal; Lane 3: digested plasmid with rsaA C-terminal and esp; Lanes 4-8: digested plasmids from various colonies with esp.

2011.06.12-2011.06.18

20110614_PCR135 20110614_PCR246 20110617_Pro-esp-stop-rsaA 20110617_rsaA%26esp
Gel 1 of 2. Lane 1: ladder; lanes 2-4: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA C-terminal that had been digested 20110606; lane 5: PCR product of plasmid containing esp; lanes 6-8: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA C-terminal PCR product from 20110605. Only lane 6 shows successful ligation of esp and rsaA C-terminal. Gel 2 of 2. Lane 1: ladder; lanes 2-4: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA C-terminal that had been digested 20110606; lane 5: PCR product of plasmid containing esp; lanes 6-8: PCR product using VF2 and VR of plasmid containing esp ligated with rsaA C-terminal PCR product from 20110605. None of these show successful ligation. 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 esp + rsaA 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. Gel of PCR product of esp and rsaA C-terminal using new primers. Lane 1: ladder; lanes 2,3: PCR product of rsaA from chromosomal Caulobacter DNA; lane 4: PCR product of esp using new primers from chromosomal S. epidermidis DNA.

2011.06.19-2011.06.25

20110621 espAndCombo 20110621 Promoter-esp-Stop-rsaA-pMR10 20110621 rsaA 20110623_esp%2BrsaA_135
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. 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. 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. 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.
20110623_esp%2BrsaA_246 20110624_PrsaA_esp_rsaA 20110624_Pxyl_esp_rsaA 20110624_BBa_K081005_esp_rsaA
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. 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. 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. 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.

2011.06.26-2011.07.02

20110627_esprsaA%2Bpromoters_1 20110627_esprsaA%2Bpromoters_2 20110629_BBa_K081005 20110629_PromoterInserts1
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). 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. 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. 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.
20110629_PromoterInserts_PrsaA 20110629_PromoterInserts_Both 20110630_espORcombo 20110701_combo1
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. 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. 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. 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.
20110701_combo2
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.

2011.07.03-2011.07.09

20110704_combo 20110705_notCombo 20110705_Tests 20110705_TestGelExtract
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. 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. 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. 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.
20110706_espGelExtractPrep 20110706_rsaAGelExtractPrep 20110706_MoreColonies 20110706_MoreColoniesFollowup
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. 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. 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. 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).
20110707_GXandPCR_combo 20110708_BBaGel 20110708_Pxyl_PrsaA_combo 20110709_MorePromoter_combo_1
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. 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. 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. 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.
20110709_MorePromoter_combo_2
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.

2011.07.10-2011.07.16

20110713_BD1%2BpMR10_DRDX 20110713_optespC3_optespIDTdF 20110714_optespIDT
Gel of colony PCR of transformants that should carry BBa_K081005, WT esp, and rsaA in pMR10, Pxyl, WT esp, and rsaA in pSB1C3, or PrsaA, WT esp, and rsaA in pSB1C3. Lane 1: ladder; lanes 2-5: BBa_K081005, WT esp, and rsaA in pMR10; lanes 6,7: Pxyl, WT esp, and rsaA in pSB1C3; lanes 8,9: PrsaA, WT esp, and rsaA in pSB1C3. Lane 1: ladder; lanes 2-5: colony PCR of transformants that should carry the optimized esp in pSB1C3; lane 6: follow up digest of miniprep of transformant that may carry optimized esp in IDTSMART-AMP. None of these colonies were successful. Lane 1: ladder; lanes 2-9: transformants that should carry optimized esp in IDTSMART-AMP; lane 10: confirmation of digest of miniprep of strain carrying BBa_K081005, WT esp, and rsaA in pMR10. Lanes 4, 7, and 9 show successful transformation.

2011.07.17-2011.07.23

20110718_optespC3_optespPrsaAC3 20110718_optespC3_optespPxylC3 20110718_optesprsaAGXIDTSMART_optesprsaAC3 20110720_PrsaA%2Boesp%2BrsaA_oesp%2BrsaAC3
Lane 1: ladder; lanes 2-4: transformants that should carry optimized esp in pSB1C3; lanes 5-10: transformants that should carry optimized esp behind PrsaA. Lane 9 has the desired gene sequence. Lane 1: ladder; lanes 2-4: transformants that should carry optimized esp in pSB1C3; lanes 5-10: transformants that should carry optimized esp behind Pxyl. Lanes 5 and 7 have the desired gene sequence. Lane 1: ladder; lanes 2-7: transformants that should carry optimized esp with rsaA from gel extraction in IDTSMART-AMP. Lanes 3, 4, and 6 have the desired gene sequence. PCR of transformants that should carry PrsaA, optimized esp, and rsaA or optimized esp with rsaA in pSB1C3. Lane 1: ladder; lanes 2-7:combo with PrsaA; lanes 8-10: combo without promoter. Lanes 3, 8, and 10 all have the desired inserts.
20110720_Pxyl%2Boesp%2BrsaA_oesp%2BrsaAC3 20110722_Bba%2Boesp 20110723_BBa%2Boesp 20110723_PrsaA%2Boesp%2BrsaA%2BpMR10
Lane 1: ladder; lanes 2-7: PCR of transformants that should carry Pxyl, optimized esp, and rsaA; lanes 8-10: transformants that should carry optimized esp with rsaA in pSB1C3. Lane 1: ladder; lanes 2-7: transformants that should carry BBa_K081005 and optimized esp. Gel of digests of miniprepped overnight liquid culture of cells that should carry BBa_K081005 and optimized esp. Lane 1: ladder; lanes 2-10: digests. Lane 1: ladder; lanes 2-7: PCR of transformants that should carry PrsaA, optimized esp, and rsaA in pMR10. Lane 4 was successful.
20110723_Pxyl%2Boesp%2BrsaA%2BpMR10
Lane 1: ladder; lanes 2-7: PCR of transformants that should carry Pxyl, optimized esp, and rsaA in pMR10.

2011.07.24-2011.07.31

20110725_Pxyl%2Boesp%2BrsaA%2BpMR10 20110727_BBa%2Boesp%2BrsaA 20110728_BD1_Caulobacter 20110729_BBa%2Boesp%2BrsaA%2BpMR10_C3
Lane 1: ladder; lanes 2-8: digests of transformants that should contain Pxyl, optimized esp, and rsaA. Lane 1: ladder; lanes 2-8: digests of miniprep of transformants that should carry BBa_K081005, optimized esp, and rsaA. No success. Lane 1: ladder; lanes 2-5: PCR of Caulobacter colonies believed to carry BBa_K081005, WT esp, and rsaA. All passed. Lane 1: ladder; lanes 2,3: PCR of transformants that should carry BBa_K081005, optimized esp, and rsaA in pMR10; lanes 4-10: PCR of transformants that should carry BBa_K081005, optimized esp, and rsaA in pSB1C3. No success.

2011.08.01-2011.08.07

20110801_BBa%2Boesp%2BrsaA%2BpMR10
Lane 1: ladder; lanes 2-10: transformants that should contain BBa_K081005, optimized esp, and rsaA. Lanes 2 and 8 show success.

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