Minnesota/17 July 2008

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|1. '''Plasmid prep dual promoters:''' (1) Tet & P22mnt, (2) LacI & LAMBDAcI.  
|1. '''Plasmid prep dual promoters:''' (1) Tet & P22mnt, (2) LacI & LAMBDAcI.  
|-
|-
-
|2. '''Double digest of all ligations:''' from previous days.
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|2. '''Model:''' Figure out why have such a low GFP output when run model.  
|-
|-
|3. '''Sequence L5, L6-L9'''
|3. '''Sequence L5, L6-L9'''
|-
|-
-
|4. '''Re-transform RFP, TetR promoter, terminator''' because no cell growth on plates.  
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|4. '''Re-transform RFP, TetR promoter, terminator, and MCherry''' because no cell growth on plates. Place in 2mL LB cultures, allow growth in incubator @37C for 2 hours. Plate samples on Ampicillin resistant plates. Allow growth O/N.  
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|5. '''Discuss problems with sequences'''
|5. '''Discuss problems with sequences'''
|-
|-
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|6. '''Model:''' ?? Why GFP output is low???
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|6. '''Double digest of all ligations:''' from previous days. Do a double digest to cut the components linearly, which will then be able to run through a gel to check if correct DNA
 +
is present. Follow the table below:
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{|
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{|border="1" align="left"
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|-
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!| Parts !! 10x Buffer !! BSA !! H20 !! DNA !! RE 1 !! RE 2
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|-
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|L2a, LAMBDAcI:Term ||5.0uL ||0.5uL ||32.5uL ||10.0uL ||EcoRI || Xba1
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|-
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|L2b, LAMBDAcI:Term ||5.0uL ||0.5uL ||32.5uL ||10.0uL ||EcoRI || Xba1
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|-
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|L3i, Pro:LAMBDAcI ||5.0uL ||0.5uL ||32.5uL ||10.0uL || Pst1 || Spe1
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|-
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|L3j, Pro:LAMBDAcI ||5.0uL ||0.5uL ||32.5uL ||10.0uL || Pst1 || Spe1
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|-
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|L4b, LAMBDAcI:Term ||5.0uL ||0.5uL ||40.5uL || 2.0uL || EcoRI || Xba1
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|-
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|L4c, LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL || 3.0uL || EcoRI || Xba1
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|-
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|L4d, LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL || 3.0uL || EcoRI || Xba1
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|-
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|L4e, LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL || 3.0uL || EcoRI || Xba1
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|-
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|L4g, LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL ||3.0uL || EcoRI || Xba1
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|-
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|L4h, LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL ||3.0uL || EcoRI || Xba1
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|-
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|L4i, LAMBDAcI:Term ||5.0uL ||0.5uL ||37.5uL ||5.0uL || EcoRI || Xba1
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|-
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|L4j, LAMBDAcI:Term ||5.0uL ||0.5uL ||32.5uL ||10.0uL || EcoRI || Xba1
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|-
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|L5c, GFP:Term ||5.0uL ||0.5uL ||40.5uL ||2.0uL || EcoRI || Xba1
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|-
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|L6a, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL ||3.0uL || EcoRI || Xba1
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|-
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|L6b, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||41.5uL || 1.0uL ||EcoRI || Xba1
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|-
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|L6e, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL ||3.0uL || EcoRI || Xba1
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|-
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|L6d, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL ||3.0uL || EcoRI || Xba1
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|-
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|L7a, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||38.5uL ||4.0uL ||EcoRI || Xba1
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|-
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|L7b, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL ||3.0uL ||EcoRI || Xba1
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|-
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|L7d, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||37.5uL || 5.0uL || EcoRI || Xba1
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|-
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|L8a, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||39.5uL ||3.0uL || EcoRI || Xba1
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|-
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|L9a, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||41.5uL ||1.0uL || EcoRI || Xba1
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|-
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|L9c, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||40.5uL ||2.0uL || EcoRI || Xba1
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|-
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|L9d, Pro:LAMBDAcI:Term ||5.0uL ||0.5uL ||41.5uL ||1.0uL || EcoRI || Xba1
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|}
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|-
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|7. '''Gel Electrophoresis:''' Performed on double digest reaction from above. Refer to picture below for results.
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|-
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|[[Image:7.17.08gel.jpg|thumb|center|600px|Gel from 07-17-2008]]
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|-
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|''Left to Right:'' Ladder, --, L9E, L9C, L9A, L8A, L7D, L7B, L7A, L6E, L6D, L6B, L6A, L5C, --, Ladder, L4j, L4i, L4h, L4g, L4d, L4e, L4c, L4b, L3j, L3i, L2b, L2a, --, Ladder
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|-
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|}

Latest revision as of 20:55, 5 August 2008

Back to Notebook Home
Go to Previous Day (July 16)Go to Next Day (July 18)
1. Plasmid prep dual promoters: (1) Tet & P22mnt, (2) LacI & LAMBDAcI.
2. Model: Figure out why have such a low GFP output when run model.
3. Sequence L5, L6-L9
4. Re-transform RFP, TetR promoter, terminator, and MCherry because no cell growth on plates. Place in 2mL LB cultures, allow growth in incubator @37C for 2 hours. Plate samples on Ampicillin resistant plates. Allow growth O/N.
5. Discuss problems with sequences
6. Double digest of all ligations: from previous days. Do a double digest to cut the components linearly, which will then be able to run through a gel to check if correct DNA

is present. Follow the table below:

Parts 10x Buffer BSA H20 DNA RE 1 RE 2
L2a, LAMBDAcI:Term 5.0uL 0.5uL 32.5uL 10.0uL EcoRI Xba1
L2b, LAMBDAcI:Term 5.0uL 0.5uL 32.5uL 10.0uL EcoRI Xba1
L3i, Pro:LAMBDAcI 5.0uL 0.5uL 32.5uL 10.0uL Pst1 Spe1
L3j, Pro:LAMBDAcI 5.0uL 0.5uL 32.5uL 10.0uL Pst1 Spe1
L4b, LAMBDAcI:Term 5.0uL 0.5uL 40.5uL 2.0uL EcoRI Xba1
L4c, LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L4d, LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L4e, LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L4g, LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L4h, LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L4i, LAMBDAcI:Term 5.0uL 0.5uL 37.5uL 5.0uL EcoRI Xba1
L4j, LAMBDAcI:Term 5.0uL 0.5uL 32.5uL 10.0uL EcoRI Xba1
L5c, GFP:Term 5.0uL 0.5uL 40.5uL 2.0uL EcoRI Xba1
L6a, Pro:LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L6b, Pro:LAMBDAcI:Term 5.0uL 0.5uL 41.5uL 1.0uL EcoRI Xba1
L6e, Pro:LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L6d, Pro:LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L7a, Pro:LAMBDAcI:Term 5.0uL 0.5uL 38.5uL 4.0uL EcoRI Xba1
L7b, Pro:LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L7d, Pro:LAMBDAcI:Term 5.0uL 0.5uL 37.5uL 5.0uL EcoRI Xba1
L8a, Pro:LAMBDAcI:Term 5.0uL 0.5uL 39.5uL 3.0uL EcoRI Xba1
L9a, Pro:LAMBDAcI:Term 5.0uL 0.5uL 41.5uL 1.0uL EcoRI Xba1
L9c, Pro:LAMBDAcI:Term 5.0uL 0.5uL 40.5uL 2.0uL EcoRI Xba1
L9d, Pro:LAMBDAcI:Term 5.0uL 0.5uL 41.5uL 1.0uL EcoRI Xba1


7. Gel Electrophoresis: Performed on double digest reaction from above. Refer to picture below for results.
Gel from 07-17-2008
Left to Right: Ladder, --, L9E, L9C, L9A, L8A, L7D, L7B, L7A, L6E, L6D, L6B, L6A, L5C, --, Ladder, L4j, L4i, L4h, L4g, L4d, L4e, L4c, L4b, L3j, L3i, L2b, L2a, --, Ladder
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