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User:University of Washington/27 June 2008
From 2008.igem.org
(New page: == Conjugation ==<br\> ''Mini Prep''<br\> ·R0042 (LuxR promoter) ''Glycerol''<br\> ·R0042 (LuxR promoter)) |
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| - | == Conjugation == | + | == Yeast Shuttle Plasmid == |
| + | |||
| + | - 1.5 mL 15% glycerol stocks of the overnight BY4741 yeast growth cultures were made by combining 563 uL of 40% glycerol and 937 uL yeast culture, then stored at -80 degrees Celsius. | ||
| + | |||
| + | == Conjugation == | ||
''Mini Prep''<br\> | ''Mini Prep''<br\> | ||
| - | + | ·R0062 (LuxR promoter) | |
''Glycerol''<br\> | ''Glycerol''<br\> | ||
| - | + | ·"" | |
| + | |||
| + | ''Sequencing''<br\> | ||
| + | ·"" | ||
| + | |||
| + | [[UW Glycerol Stocks]] | ||
| + | |||
| + | == LuxR from AraC and TetR == | ||
| + | |||
| + | - Made glycerol stock | ||
| + | |||
| + | - Did mini Prep | ||
| + | |||
| + | - Submitted DNA for sequencing reaction and analysis (only for AraC) | ||
| + | |||
| + | == LuxR from pLac == | ||
| + | |||
| + | - I0462 was again transformed into MG strain again, and plated on AMP. | ||
| + | |||
| + | == BioBrick Promoter Measurements == | ||
| + | |||
| + | - A strain of TOP10 E. coli was obtained and thawed on ice, along with a strain which had been previously transformed (at MIT) with the fully-assembled reference promoter-RBS-GFP gene-backbone plasmid (I20260). | ||
| + | |||
| + | - 40 uL aliquots of the untransformed cells were transferred into five different tubes. A 40 uL aliquot of the already-transformed cells was similarly transferred into its own tube. | ||
| + | |||
| + | - 2 uL each of plasmid solutions I20260, J23150, J23151, and J23102 were added to four separate aliquots of TOP10 cells. | ||
| + | |||
| + | - The TOP10-pDNA mixtures, the pretransformed TOP10 cells, and the remaining negative control with only untransformed TOP10 cells were incubated on ice for 5 minutes, then incubated in a water bath at 42 degrees Celsius for 30 seconds, then transferred back to ice. | ||
| + | |||
| + | - 250 uL of SOC media was added to each of the TOP10 tubes, then incubated at 37 degrees Celsius on a rotator for 1 hour. | ||
| + | |||
| + | - 100 uL of the each of the transformed cells were placed on Kanamycin plates, then left at room temperature over the weekend to allow for slow growth. | ||
| + | |||
| + | |||
| + | |||
| + | ---- | ||
| + | |||
| + | [[Team:University_of_Washington/Notebook#Notebook]] | ||
Latest revision as of 22:53, 2 July 2008
Contents |
Yeast Shuttle Plasmid
- 1.5 mL 15% glycerol stocks of the overnight BY4741 yeast growth cultures were made by combining 563 uL of 40% glycerol and 937 uL yeast culture, then stored at -80 degrees Celsius.
Conjugation
Mini Prep
·R0062 (LuxR promoter)
Glycerol
·""
Sequencing
·""
LuxR from AraC and TetR
- Made glycerol stock
- Did mini Prep
- Submitted DNA for sequencing reaction and analysis (only for AraC)
LuxR from pLac
- I0462 was again transformed into MG strain again, and plated on AMP.
BioBrick Promoter Measurements
- A strain of TOP10 E. coli was obtained and thawed on ice, along with a strain which had been previously transformed (at MIT) with the fully-assembled reference promoter-RBS-GFP gene-backbone plasmid (I20260).
- 40 uL aliquots of the untransformed cells were transferred into five different tubes. A 40 uL aliquot of the already-transformed cells was similarly transferred into its own tube.
- 2 uL each of plasmid solutions I20260, J23150, J23151, and J23102 were added to four separate aliquots of TOP10 cells.
- The TOP10-pDNA mixtures, the pretransformed TOP10 cells, and the remaining negative control with only untransformed TOP10 cells were incubated on ice for 5 minutes, then incubated in a water bath at 42 degrees Celsius for 30 seconds, then transferred back to ice.
- 250 uL of SOC media was added to each of the TOP10 tubes, then incubated at 37 degrees Celsius on a rotator for 1 hour.
- 100 uL of the each of the transformed cells were placed on Kanamycin plates, then left at room temperature over the weekend to allow for slow growth.
Team:University_of_Washington/Notebook#Notebook
