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Team:KULeuven/8 August 2008
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* We also started to construct the T7 polymerase with UmuD tag, using PCR. | * We also started to construct the T7 polymerase with UmuD tag, using PCR. | ||
* We electroporated some ligated parts: R0084 + B0032, R0062 + B0032, R0084 + J23022 and C0012 + B0015 | * We electroporated some ligated parts: R0084 + B0032, R0062 + B0032, R0084 + J23022 and C0012 + B0015 | ||
| - | * We made a liquid culture of parts B0032, C0060, C0062, P1010, ?. On Monday, we can | + | * We made a liquid culture of parts B0032, C0060, C0062, P1010, ?. On Monday, we can MiniPrep them. |
* We also made a liquid culture of Top10 cells to make new electrocompetent cells. | * We also made a liquid culture of Top10 cells to make new electrocompetent cells. | ||
Revision as of 17:49, 10 August 2008
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Contents |
Lab Work
Wet Lab
- We tested C0040 + B0015, P1010 + B0015, C0060 + B0015 and J23100 + B0032 to see if the ligation succeed. The results were disappointing. None of them were properly ligated.
- We made some MiniPreps of some other ligated parts (E0022 + B0015, J23109 + J23032, I712074 + J23082, GFP-LVA + B0015, GFP-LVA + pSB1A2 and C0062 + B0015). After that, we did a PCR using the VR/VF2 primers to see whether these ligations worked or not. E0022 + B0015, GFP-LVA + pSB1A2 and C0062 + B0015 were OK. J23109 + J23032, I712074 + J23082 and GFP-LVA + B0015 gave mixed results. Some of the colonies contained ligated parts, others did not. So we have to try to isolate the right colonies.
- We also started to construct the T7 polymerase with UmuD tag, using PCR.
- We electroporated some ligated parts: R0084 + B0032, R0062 + B0032, R0084 + J23022 and C0012 + B0015
- We made a liquid culture of parts B0032, C0060, C0062, P1010, ?. On Monday, we can MiniPrep them.
- We also made a liquid culture of Top10 cells to make new electrocompetent cells.
Dry Lab
Hybrid promoter in front of ccdB is not enough because the timer will have started. So the safest way might be to use antisense LuxI (leaky thouh it is) and the hybrid promoter. If it's too much work/costs, just antisense LuxI maybe.
The following parts will have to be on a higher copy number plasmid (preferably a pSB5X#): antisense LuxI, the ribokey and the 434 without the LVA tag.
