Team:Michigan/Oil Sands October
From 2010.igem.org
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''Marcus'' | ''Marcus'' | ||
- | Made 10 Tet and 10 Cm plates. Also, Nick and I transformed DH5alpha comp cells from - | + | Made 10 Tet and 10 Cm plates. Also, Nick and I transformed DH5alpha comp cells from -80ºC iGEM box with extra pSB1AT3-J23100-Flu ligation Ben and Kevin made yesterday. Transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]]. We also transformed ''P. putida'' and ''P. fluorescens'' with pSB1AT3 as a test. No dilutions were made. It was noted while plating the cells that the ''Pseudomonas'' strains somehow wound up with less than 50 uL of cells. And due to the cells flocculating during storage, the ''Pseudomonas'' strains also had be vortexed to resuspend the cells. |
== 10/21/2010 == | == 10/21/2010 == | ||
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**VF2- 33.9 nmol/.05 nmol/uL= 678 uL | **VF2- 33.9 nmol/.05 nmol/uL= 678 uL | ||
**VR- 40 nmol/ .05 nmol/uL= 800 uL | **VR- 40 nmol/ .05 nmol/uL= 800 uL | ||
- | *Used a 2 min | + | *Used a 2 min extension |
- | Also, put the Cm and Tet plates from Tuesday in the | + | Also, put the Cm and Tet plates from Tuesday in the 4ºC. |
== 10/23/2010 == | == 10/23/2010 == | ||
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'''Expected Digest bands''' | '''Expected Digest bands''' | ||
- | *FimB- 638 bp | + | *FimB - 638 bp |
- | *VP130- 2376, 1925 bp | + | *VP130 - 2376, 1925 bp |
- | *pSB1AT3-Flu- 6770, 15 bp | + | *pSB1AT3-Flu - 6770, 15 bp |
The digests appear to have worked, although the ligations seem to have been incomplete (Roche Rapid Dephos & Ligation Kit). | The digests appear to have worked, although the ligations seem to have been incomplete (Roche Rapid Dephos & Ligation Kit). | ||
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*16.7 uL UP H2O | *16.7 uL UP H2O | ||
- | We then did the following transformations using the [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]] and the | + | We then did the following transformations using the [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]] and the DH5α comp cells Nick made using his [[Media:Quick Transformation of E. coli Competant Cell Preperation.pdf| Competent Cell Preparation for Quick Transformation of E. coli]] protocol: |
Transformation (Plate Resistance): | Transformation (Plate Resistance): | ||
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Ligation: | Ligation: | ||
- | *pSB1C3- 4.572 fmol/uL | + | *pSB1C3 - 4.572 fmol/uL |
- | *VP130- 8.065 fmol/uL | + | *VP130 - 8.065 fmol/uL |
- | *FimB- 24.2 fmol/uL | + | *FimB - 24.2 fmol/uL |
VP130 Ligation Mix: | VP130 Ligation Mix: | ||
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Incubation: | Incubation: | ||
- | *10 mins @ 22. | + | *10 mins @ 22.5ºC |
- | *10 mins @ | + | *10 mins @ 65ºC |
Ligations were transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]]. | Ligations were transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]]. |
Latest revision as of 01:08, 27 October 2010