Minnesota/18 July 2008

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Latest revision as of 18:40, 21 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 Restriction 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. Follow the table below:

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

4. Ligate Digested products: Ligated -

a. Lac/LAMBDAcI promoter + GFP:Term + BaseVector

b. TetR/p22mnt promoter + YFP + BaseVector

Parts	10x Buffer	H20	Prom	Reporter	T4 Ligase
(a) Lac/LAMBDAcI : GFP : Term	3.0uL	6.0uL	15.0uL	5.0uL	1.0uL
(b) Lac/LAMBDAcI : GFP : Term	3.0uL	6.0uL	15.0uL	5.0uL	1.0uL
TetR/p22mnt : YFP	3.0uL	6.0uL	15.0uL	5.0uL	1.0uL

5. 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.

6. Sequencing Rxns: Ran the parts through a gel on 07-17-08, we selected the lanes with the best outcomes. Send the following into Biomedical Genomics Center to be sequenced -

Part	Primer	DNA	H20	KJL
(a) L3i	3.2uL, LAMBDAcI F	5.0uL	3.8uL	142
(b) L3i	3.2uL, BaseVec. F	5.0uL	3.8uL	143
(a) L3j	3.2uL, LAMBDAcI F	5.0uL	3.8uL	144
(b) L3j	3.2uL, BaseVec. F	5.0uL	3.8uL	145
(a) L4b	3.2uL, LAMBDAcI F	2.4uL	4.6 uL	146
(b) L4b	3.2uL, BaseVec. F	2.4uL	4.6uL	147
(a) L4i	3.2uL, LAMBDAcI F	6.0uL	2.8uL	148
(b) L4i	3.2uL, BaseVec. F	6.0uL	2.8uL	149
(a) L5c	3.2uL, LAMBDAcI F	3.5uL	5.3uL	150
(b) L5c	3.2uL, BaseVec. F	3.5uL	5.3uL	151
(a) L6a	3.2uL, LAMBDAcI F	5.0uL	3.8uL	152
(b) L6a	3.2uL, BaseVec. F	5.0uL	3.8uL	153
(a) L6b	3.2uL, LAMBDAcI F	2.2uL	6.6uL	154
(b) L6b	3.2uL, BaseVec. F	2.2uL	6.6uL	155
(a) L6d	3.2uL, LAMBDAcI F	5.0uL	3.8uL	156
(b) L6d	3.2uL, BaseVec. F	5.0uL	3.8uL	157
(a) L6e	3.2uL, LAMBDAcI F	5.0uL	3.8uL	158
(b) L6e	3.2uL, BaseVec. F	5.0uL	3.8uL	159
(a) L7a	3.2uL, LAMBDAcI F	6.0uL	2.8uL	160
(b) L7a	3.2uL, BaseVec. F	6.0uL	2.8uL	161
(a) L7b	3.2uL, LAMBDAcI F	5.0uL	3.8uL	162
(b) L7b	3.2uL, BaseVec. F	5.0uL	3.8uL	163
(a) L7d	3.2uL, LAMBDAcI	5.0uL	3.8uL	164
(b) L7d	3.2uL, BaseVec. F	5.0uL	3.8uL	165
(a) L8a	3.2uL, LAMBDAcI	5.0uL	3.8uL	166
(b) L8a	3.2uL, BaseVec. F	5.0uL	3.8uL	167

@@ Line 59: / Line 59: @@
 |'''5. 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.
 |-
-|'''6. Sequencing Rxns:''' Ran the parts through a gel on 07-17-08, we selected the lanes with the best outcomes. Send into Biomedical Genomics Center to be sequenced -
+|'''6. Sequencing Rxns:''' Ran the parts through a gel on 07-17-08, we selected the lanes with the best outcomes. Send the following into Biomedical Genomics Center to be sequenced -
 {|border="1" align="left"