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User:University of Washington/1 August 2008
From 2008.igem.org
(→LuxR from AraC and TetR) |
(→Cure MG1655Z1) |
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| + | - After 2:45 hours incubation, 30 ul of cut DNA was used to run on gel. All, except control, showed two bands: one between 4-5 kb, the other between 8-10 kb. (Expected pCD26 9.5 kb, pACYC184 4.2 kb) Possible conclusion was the strain contained plasmid pACYC184 that produce LuxR as mentioned in the literature. | ||
| + | |||
| + | - 8 colonies were selected from second Tsy plate and inoculated on LB + Kan plate to check if we had lost the promoter plasmid(pCD26 with KAN resistance). | ||
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Back to [[Team:University_of_Washington/Notebook#Notebook]] | Back to [[Team:University_of_Washington/Notebook#Notebook]] | ||
Revision as of 00:01, 2 August 2008
LuxR from AraC and TetR
- BBa_P1010 (contains death gene) grew in both XL1-Blue and DB3.1 which was weird. XL1-Blue didn't have death resistance, so the cell shouldn't grow. Will miniprep and sequence the part P1010. But will use the vector plasmid containing Amp resistance gene from the part nevertheless.
- Obtained P1010 on pSB3K3 and pSB1AC3 from Brandi by inoculated the strain on selective plates.
Cure MG1655Z1
- Restriction digested the Elowitz's plasmid from original plate and stock plate with XbaI and BamHI (both cut both plasmid pACYC184 and pCD26 once)
| Reagents | Ori + XbaI | Stock + XbaI | Ori + BamHI | Stock + BamHI | Control |
|---|---|---|---|---|---|
| dH2O | 34 ul | 34 ul | 39 ul | 39 ul | 39.5 ul |
| Buffer | 5 ul NEB2 | 5 ul NEB2 | 5 ul NEB3 | 5 ul NEB3 | 5 ul NEB3 |
| BSA | 0.5 ul | 0.5 ul | 0.5 ul | 0.5 ul | 0.5 ul |
| DNA | 10 ul | 10 ul | 5 ul | 5 ul | 5 ul |
| vortex | |||||
| Enzyme | 0.5 ul XbaI | 0.5 ul XbaI | 0.5 ul BamHI | 0.5 ul BamHI | - |
| centrifuge and incubate 37 degree Celsius | |||||
- After 2:45 hours incubation, 30 ul of cut DNA was used to run on gel. All, except control, showed two bands: one between 4-5 kb, the other between 8-10 kb. (Expected pCD26 9.5 kb, pACYC184 4.2 kb) Possible conclusion was the strain contained plasmid pACYC184 that produce LuxR as mentioned in the literature.
- 8 colonies were selected from second Tsy plate and inoculated on LB + Kan plate to check if we had lost the promoter plasmid(pCD26 with KAN resistance).
Back to Team:University_of_Washington/Notebook#Notebook
