User:University of Washington/27 June 2008

From 2008.igem.org

(Difference between revisions)
(New page: == Conjugation ==<br\> ''Mini Prep''<br\> ·R0042 (LuxR promoter) ''Glycerol''<br\> ·R0042 (LuxR promoter))
 
(12 intermediate revisions not shown)
Line 1: Line 1:
-
== Conjugation ==<br\>
+
== 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\>
-
·R0042 (LuxR promoter)
+
·R0062 (LuxR promoter)
''Glycerol''<br\>
''Glycerol''<br\>
-
·R0042 (LuxR promoter)
+
·""
 +
 
 +
''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
·""

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