Team:Grinnell/Notebook/Gels/DspB

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Lane 1: ladder; lanes 2-5: transformants that should carry P<sub>rsaA</sub>, optimized <i>dspB</i>, and <i>rsaA</i>; lanes 6-9: transformants that should carry P<sub>xyl</sub>, optimized <i>dspB</i>, and <i>rsaA</i>; lane 10: standard optimized <I>dspB</I> from PCR. None of these transformants showed success.
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Figure 9: Optimized <i>dspB + <i> rsaA</i> with P<sub>xyl</sub> and P<sub>rsaA</sub>.  Lane 1: ladder; lanes 2-5: P<sub>rsaA</sub> + optimized <i>dspB</i> + <i>rsaA</i> colonies 1-4; lanes 6-9: P<sub>xyl</sub> + optimized <i>dspB</i> + <i>rsaA</i> colonis 1-4; lane 10: standard optimized <I>dspB</I> + <i>rsaA</i>. P<sub>rsaA</sub> + optimized <i>dspB</i> + <i>rsaA</i> colony 3 shows the expected size.
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Revision as of 19:50, 27 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. Figure 4: Test of more WT dspB with Pxyl colonies. Lane 1: ladder; lanes 2-5: WT dspB with Pxyl colonies 5-8; 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
Figure 5: Optimized dspB transformed into pSB1C3 colonies A-E. Lane 1: ladder; lanes 2-5: optimized dspB colonies A-E; lanes 6-9: optimized esp. Colony D shows the expected size. Figure 6: More colonies of WT dspB with Pxyl promoter. Lanes 1-4: colonies 9, 10, 12, and 15; lane 5: dspB WT standard from PCR. Figure 7: The rest of the PCR product of colony 10 from Figure 6 in preparation for gel extraction of WT dspB behind Pxyl. Lane 1: ladder; lanes 2,3: WT dspB with Pxyl. Figure 8: 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
Figure 9: Optimized dspB + rsaA with Pxyl and PrsaA. Lane 1: ladder; lanes 2-5: PrsaA + optimized dspB + rsaA colonies 1-4; lanes 6-9: Pxyl + optimized dspB + rsaA colonis 1-4; lane 10: standard optimized dspB + rsaA. PrsaA + optimized dspB + rsaA colony 3 shows the expected size. 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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