Team:Grinnell/Notebook/Gels/DspB

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Gel proving the successful transformation of WT <i>dspB</i> from UBC into <i>E. coli</i> Top10. Lane 1: ladder; lanes 2-4: digests (EcoRI and PstI) of miniprep DNA from overnights of transformant colonies 1-3. There is a clear band at the appropriate size for <i>dspB</i> in all of the experimental lanes.
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Figure 1: Gel proving the successful transformation of WT <i>dspB</i> from UBC into <i>E. coli</i> Top10. Lane 1: ladder; lanes 2-4: digests (EcoRI and PstI) of miniprep DNA from overnights of transformant colonies 1-3. There is a clear band at the appropriate size for <i>dspB</i> in all of the experimental lanes.
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Gel of transformants that should contain both WT <i>dspB</i> and a promoter insert. Lane 1: ladder; lanes 2-4: WT <i>dspB</i> with P<sub>rsaA</sub> colonies 1-4; lanes 5-7: WT <i>dspB</i> with P<sub>xyl</sub> colonies 1-4; lane 8: standard <i>dspB</i> with no promoter.
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Figure 2: Gel of transformants that should contain both WT <i>dspB</i> and a promoter insert. Lane 1: ladder; lanes 2-4: WT <i>dspB</i> with P<sub>rsaA</sub> colonies 1-4; lanes 5-7: WT <i>dspB</i> with P<sub>xyl</sub> colonies 1-4; lane 8: standard <i>dspB</i> with no promoter.
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Gel showing <i>dspB</i> with various promoter inserts as contrasted to a standard <i>dspB</i> with no promoter. Lane 1: dspB (WT) + P<sub>rsaA</sub>; lane 2: with P<sub>xyl</sub>; lanes 3,4: with BBa_K081005; lane 5: standard <i>dspB</i>; lane 6: ladder. Not all transformants show successful insertion of the desired promoter, but it is clear which were successful when compared to the standard <i>dspB</i>.
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Figure 3: Follow up to Figure 2.  Gel showing WT <i>dspB</i> with various promoter inserts as contrasted to a standard <i>dspB</i> with no promoter. Lane 1: WT <i>dspB</i> + P<sub>rsaA</sub> colony 3; lane 2: with P<sub>xyl</sub> colony 3; lanes 3,4: with BBa_K081005 colonies 3 and 4; lane 5: standard WT <i>dspB</i>; lane 6: ladder.
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Revision as of 20:24, 26 July 2011

Grinnell Menubar

Gels for DspB

2011.07.03-2011.07.09

20110707_UBC_dspB_transformation_results_colonies_1_2_3 20110708_dspB%2BPrsaA_Pxyl 20110708_dspB%2BpromotersVSdspB_std 20110709dspB%2Bpxyl_Colonies5-8
Figure 1: Gel proving the successful transformation of WT dspB from UBC into E. coli Top10. Lane 1: ladder; lanes 2-4: digests (EcoRI and PstI) of miniprep DNA from overnights of transformant colonies 1-3. There is a clear band at the appropriate size for dspB in all of the experimental lanes. Figure 2: Gel of transformants that should contain both WT dspB and a promoter insert. Lane 1: ladder; lanes 2-4: WT dspB with PrsaA colonies 1-4; lanes 5-7: WT dspB with Pxyl colonies 1-4; lane 8: standard dspB with no promoter. Figure 3: Follow up to Figure 2. Gel showing WT dspB with various promoter inserts as contrasted to a standard dspB with no promoter. Lane 1: WT dspB + PrsaA colony 3; lane 2: with Pxyl colony 3; lanes 3,4: with BBa_K081005 colonies 3 and 4; lane 5: standard WT dspB; lane 6: ladder. Gel of more transformants of dspB with Pxyl. Lane 1: ladder; lanes 2-5: transformants; lane 6: standard dspB with no promoter.


2011.07.10-2011.07.16

20110710_optGenes%2BC3 20110711_c9_10_12_15_from20110707transf-Pxyl%2BdspBWT 20110712_dspB%2BPxyl_forGX 20110712_optdspB%2BrsaAc1-4_optdspB
Gel of transformants with synthesized dspB and esp with codons optimized for expression in Caulobacter after ligation into pSB1C3. Lane 1: ladder; lanes 2-5: optimized dspB; lanes 6-9: optimized esp. Lane 4 shows successful insertion of dspB into pSB1C3, but none of the optimized esp transformants show the desired insert. Gel of colony PCR of transformants that should carry WT dspB behind the Pxyl promoter. Lanes 1-4: colonies 9, 10, 12, and 15 from transformation plate; lane 5: dspB WT standard from PCR. Gel for extraction of WT dspB behind Pxyl. Lane 1: ladder; lanes 2,3: miniprep digest. Gel of colony PCR of transformants carrying optimized dspB and rsaA C-terminal. Lane 1: ladder; lanes 2-5: transformation colonies 1-4; lane 6: dspB standard from PCR of WT dspB.
20110714_optdspB%2BrsaA%2BPrsaA_OR_Pxyl 20110714_optdspB%2BrsaA%2BPxyl_c5-8 20110714_WTdspB%2BPxyl 20110715_BBa%2BoptdspB%2BrsaA
Lane 1: ladder; lanes 2-5: transformants that should carry PrsaA, optimized dspB, and rsaA; lanes 6-9: transformants that should carry Pxyl, optimized dspB, and rsaA; lane 10: standard optimized dspB from PCR. None of these transformants showed success. Lane 1: ladder; lanes 2-5: transformants that should carry Pxyl, optimized dspB, and rsaA; lane 6: standard optimized dspB with rsaA from PCR. None of these colonies were successful. Lane 1: standard optimized dspB with rsaA from PCR; lanes 2,3: transformants that should carry WT dspB behind Pxyl; lane 4: ladder. No successful transformations. Lane 1: ladder; lanes 2-5: transformants that should carry BBa_K081005, optimized dspB, and rsaA; lane 6: standard optimized dspB with rsaA fragment. Lanes 3 and 5 carry the desired combination of genes.
20110715_Pxyl%2BoptdspB%2BrsaA_c9-14
Lane 1: ladder; lanes 2-7: transformants that should carry Pxyl, optimized dspB, and rsaA; lane 8: standard fragment of optimized dspB with rsaA. None of the transformants carried the desired gene sequence.

2011.07.17-2011.07.23

20110718_BBa_OR_PrsaA%2BoptdspB%2BrsaA_pMR10 20110719_Pxyl%2BodspB%2BrsaA_re-do_c1,3,4,5,6_std 20110720_Pxyl%2BodspB%2BrsaA_re-do_c7-14_WTdspB_c5 20110722_BBa%2BodspB%2BrsaA%2BpMR10_c1,5-9
Lane 1: ladder; lanes 2-5: transformants that should carry BBa_K081005, optimized dspB, and rsaA in pMR10; lanes 6-9: transformants that should carry PrsaA, optimized dspB, and rsaA in pMR10; lane 10: standard DNA fragment of optimized dspB with rsaA. Lane 7 carries the desired gene sequence. Gel of PCR of transformants that should carry Pxyl, optimized dspB, and rsaA. Lane 1: ladder; lanes 2-6: PCR products; lane 7: standard fragment of dspB with rsaA. Gel of PCR of transformants that should carry Pxyl, optimized dspB, and rsaA. Lane 1: ladder; lanes 2-9: transformant PCR products; lane 10: digested miniprep of what should have been WT dspB, but showed up as the wrong fragment size. PCR of transformants that should carry BBa_K081005, optimized dspB, and rsaA in pMR10. Lane 1: ladder; lanes 2-7: PCR products.
20110722_Pxyl%2BodspB%2BrsaA%2BpMR10
PCR of transformants that should carry Pxyl, optimized dspB, and rsaA in pMR10. Lane 1: ladder; lanes 2-8: PCR products.

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