Minnesota/18 July 2008

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Revision as of 21:32, 18 July 2008

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1. Analyze gel results from 07-17-2008: Send in select samples from the gel for sequencing. Send in reaction mixture containing template DNA and primers that will bind to it, and thus allow to sequence. Sequencing L3i, L3j, L4b, L4i, L5c, L6a, L6b, L6d, L6e, L7a, L7b, L7d, and L8a.

2. Problem: No growth on plates with MCherry, RFP, Terminator, and TetR promoter.

Solution: Re-transform (again) paper DNA with MCherry, RFP, Term., and TetR.

3. Double Digest: Double digest dually repressed promoters; (1) Lac/LAMBDAcI, (2) TetR/p22mnt, along with reporter genes; GFP and YFP. Incubate @ 37C for 2-20 hrs. Heat inactivate for 15 mins @ 65C.

Parts	10x Buffer	BSA	H20	DNA	RE 1	RE 2
(A) Lac/LAMBDAcI Promoters	5.0uL	0.5uL	13.5uL	29.0uL	1.0uL, EcoRI	1.0uL, Spe1
(B) Lac/LAMBDAcI Promoters	5.0uL	0.5uL	9.5uL	33.0uL	1.0uL, EcoRI	1.0uL, Spe1
(A) TetR/p22mnt Promoters	5.0uL	0.5uL	34.1uL	8.4uL	1.0uL, EcoRI	1.0uL, Spe1
(B) TetR/p22mnt Promoters	5.0uL	0.5uL	34.1uL	8.4uL	1.0uL, EcoRI	1.0uL, Spe1
GFP:Term	5.0uL	0.5uL	20.5uL	22.0uL	1.0uL, EcoRI	1.0uL, Xba1

RFP will not be used because it has not been properly transformed yet. Will result in:

Lac/LAMBDAcI:GFP:Terminator

Tet/p22mnt:YFP:Terminator

5. Ligate Digested products:

4. Work on model: Model allows us to run potential reactions computationally. This will give us an idea of whether or not there will be errors in the rxns and what outputs will result.

@@ Line 13: / Line 13: @@
 ''Solution:'' Re-transform (again) paper DNA with MCherry, RFP, Term., and TetR.
 |-
-|'''3. Double Digest and Ligate:''' Double digesting then ligating dually repressed promoters; (1) Lac/LAMBDAcI, (2) TetR/p22mnt, along with reporter genes; GFP and YFP.
+|'''3. Double Digest:''' Double digest dually repressed promoters; (1) Lac/LAMBDAcI, (2) TetR/p22mnt, along with reporter genes; GFP and YFP. Incubate @ 37C for 2-20 hrs. Heat inactivate for 15 mins @ 65C.
 {|border="1" align="left"
@@ Line 19: / Line 19: @@
 !| Parts !! 10x Buffer !! BSA !! H20 !! DNA !! RE 1 !! RE 2
 |-
-|
+| (A) Lac/LAMBDAcI Promoters || 5.0uL ||0.5uL ||13.5uL ||29.0uL ||1.0uL, EcoRI || 1.0uL, Spe1
+|-
+| (B) Lac/LAMBDAcI Promoters ||5.0uL ||0.5uL ||9.5uL || 33.0uL || 1.0uL, EcoRI || 1.0uL, Spe1
+|-
+| (A) TetR/p22mnt Promoters ||5.0uL ||0.5uL ||34.1uL ||8.4uL || 1.0uL, EcoRI || 1.0uL, Spe1
+|-
+| (B) TetR/p22mnt Promoters ||5.0uL ||0.5uL ||34.1uL ||8.4uL || 1.0uL, EcoRI || 1.0uL, Spe1
+|-
+| GFP:Term ||5.0uL ||0.5uL ||20.5uL || 22.0uL ||1.0uL, EcoRI || 1.0uL, Xba1
+|}
 RFP will not be used because it has not been properly transformed yet. Will result in:
@@ Line 26: / Line 36: @@
 |-
 |Tet/p22mnt:YFP:Terminator
+|-
+|'''5. Ligate Digested products:'''
 |-
 |'''4. Work on model:''' Model allows us to run potential reactions computationally. This will give us an idea of whether or not there will be errors in the rxns and what outputs will result.
 |}