Minnesota/18 July 2008

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''Solution:'' Re-transform (again) paper DNA with MCherry, RFP, Term., and TetR.   
''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 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:
 +
 
{|border="1" align="left"
{|border="1" align="left"
Line 19: Line 20:
!| Parts !! 10x Buffer !! BSA !! H20 !! DNA !! RE 1 !! RE 2
!| 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:
+
|-
 +
|RFP will not be used because it has not been properly transformed yet. Will result in:
|-
|-
|Lac/LAMBDAcI:GFP:Terminator
|Lac/LAMBDAcI:GFP:Terminator
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|Tet/p22mnt:YFP:Terminator
|Tet/p22mnt:YFP:Terminator
|-
|-
-
|'''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.  
+
|'''4. Ligate Digested products:'''  Ligated -
 +
|-
 +
|'''a.''' Lac/LAMBDAcI promoter + GFP:Term + BaseVector
 +
|-
 +
|'''b.''' TetR/p22mnt promoter + YFP + BaseVector
 +
 
 +
 
 +
{|border="1" align="left"
 +
|-
 +
!| 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 -
 +
 
 +
 
 +
{|border="1" align="left"
 +
|-
 +
!| 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
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|-
 +
|(a) L3j || 3.2uL, LAMBDAcI F ||5.0uL || 3.8uL || 144
 +
|-
 +
|(b) L3j || 3.2uL, BaseVec. F ||5.0uL || 3.8uL || 145
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|-
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|(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
 +
|-
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|(a) L5c || 3.2uL, LAMBDAcI F || 3.5uL || 5.3uL || 150
 +
|-
 +
|(b) L5c || 3.2uL, BaseVec. F ||3.5uL || 5.3uL || 151
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|-
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|(a) L6a || 3.2uL, LAMBDAcI F || 5.0uL || 3.8uL || 152
 +
|-
 +
|(b) L6a || 3.2uL, BaseVec. F || 5.0uL || 3.8uL || 153
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|-
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|(a) L6b || 3.2uL, LAMBDAcI F || 2.2uL || 6.6uL || 154
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|-
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|(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
 +
|-
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|(a) L6e || 3.2uL, LAMBDAcI F || 5.0uL || 3.8uL || 158
 +
|-
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|(b) L6e || 3.2uL, BaseVec. F || 5.0uL || 3.8uL || 159
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|-
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|(a) L7a || 3.2uL, LAMBDAcI F || 6.0uL || 2.8uL || 160
 +
|-
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|(b) L7a || 3.2uL, BaseVec. F || 6.0uL || 2.8uL || 161
 +
|-
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|(a) L7b || 3.2uL, LAMBDAcI F || 5.0uL || 3.8uL || 162
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|-
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|(b) L7b || 3.2uL, BaseVec. F || 5.0uL || 3.8uL || 163
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|-
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|(a) L7d || 3.2uL, LAMBDAcI || 5.0uL || 3.8uL || 164
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|-
 +
|(b) L7d || 3.2uL, BaseVec. F || 5.0uL || 3.8uL || 165
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|-
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|(a) L8a || 3.2uL, LAMBDAcI || 5.0uL || 3.8uL || 166
 +
|-
 +
|(b) L8a || 3.2uL, BaseVec. F || 5.0uL || 3.8uL || 167
 +
|}
|}
|}

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
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