Minnesota/31 July 2008

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|'''[[Team:Minnesota/NotebookComparator|  Back to Notebook Home]]'''
|'''[[Team:Minnesota/NotebookComparator|  Back to Notebook Home]]'''
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|'''[[Minnesota/30 July 2008|Go to Previous Day (July 30)]]'''|| width=158|'''[[Minnesota/1 August 2008|Go to Next Day (August 1)]]'''
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|'''[[Minnesota/30 July 2008|Go to Previous Day (July 30)]]'''|| width=170|'''[[Minnesota/1 August 2008|Go to Next Day (August 1)]]'''
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|'''1. Pick Colonies from BV:Lac/LAMBDA (dual promoter and base vector):''' Picked colonies and placed BV:dualpromoters in 2mL LB cultures to incubate @ 37C for 8-12 hours.
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|'''1. Pick Colonies from BV:Lac/LAMBDA (dual promoter and base vector):''' Picked colonies and placed BV:dualpromoters in 2mL LB cultures to incubate @ 37C for 8-12 hours. (Also make one 'control' w/ 2mL LB and pipet tip but no colony on it).  
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|'''2. Plasmid Prep (1) BV:Tet/p22:RFP and (2) BV:Tetpromoter:LAMBDAcIgene:''' Using QIAmini prep guidelines - prepped 5 samples of BV:TetR/p22:RFP and 5 samples of BV:TetRpromoter:LAMBDAcIgene. ''Steps:'' Pour LB culture into tube. Spin tube and remove supernatant. Resuspend pelleted bacterial cells in 250uL Buffer P1 Resuspension buffer. Add 250uL Buffer P2 Lysis buffer and invert 4-6 times. Add 350uL of Buffer N3 Neutralization buffer and invert 4-6 times. Centrifuge for 10 minutes @ 13,000 rpm's. Put supernatants in to spin column, centrifuge for 30-60 seconds (spin column binds to DNA and everything else flows through). Discard flow through. Add 500.0uL of Buffer PB binding buffer, centrifuge for 30-60 seconds, discard flow through. Add 750.0uL Buffer PE wash buffer, centrifuge for 30-60 seconds, discard supernatants and centrifuge for an additional 30-60 seconds. Place spin column in microcentrifuge tube, and elute DNA by adding 50uL Buffer EB elution buffer (10mM Tris-Cl, pH 8.5)let stand for 5 minutes and centrifuge.     
|'''2. Plasmid Prep (1) BV:Tet/p22:RFP and (2) BV:Tetpromoter:LAMBDAcIgene:''' Using QIAmini prep guidelines - prepped 5 samples of BV:TetR/p22:RFP and 5 samples of BV:TetRpromoter:LAMBDAcIgene. ''Steps:'' Pour LB culture into tube. Spin tube and remove supernatant. Resuspend pelleted bacterial cells in 250uL Buffer P1 Resuspension buffer. Add 250uL Buffer P2 Lysis buffer and invert 4-6 times. Add 350uL of Buffer N3 Neutralization buffer and invert 4-6 times. Centrifuge for 10 minutes @ 13,000 rpm's. Put supernatants in to spin column, centrifuge for 30-60 seconds (spin column binds to DNA and everything else flows through). Discard flow through. Add 500.0uL of Buffer PB binding buffer, centrifuge for 30-60 seconds, discard flow through. Add 750.0uL Buffer PE wash buffer, centrifuge for 30-60 seconds, discard supernatants and centrifuge for an additional 30-60 seconds. Place spin column in microcentrifuge tube, and elute DNA by adding 50uL Buffer EB elution buffer (10mM Tris-Cl, pH 8.5)let stand for 5 minutes and centrifuge.     
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|'''3. Spectrophotometry:''' Spec the 10 plasmid prep samples to check for concentration of DNA.
|'''3. Spectrophotometry:''' Spec the 10 plasmid prep samples to check for concentration of DNA.
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|'''4. Double Digest:''' Digest Plasmid Prep products. After following procedure below
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{|border="1" align="left"
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!| Parts !! 10x Buffer !! BSA !! H20 !! DNA !! RE 1 !! RE 2 !! Total
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| BV + T/P + RFP (1) || 5.0uL || 0.5uL ||  2.5uL || 40.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
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| BV + T/P + RFP (2) || 5.0uL || 0.5uL ||  2.5uL || 40.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
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| BV + Tetpro. + LAMBDAcI (1) || 5.0uL || 0.5uL ||  2.5uL || 40.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
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| BV + Tetpro. + LAMBDAcI (2) || 5.0uL || 0.5uL ||  2.5uL || 40.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
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| Terminator || 5.0uL || 0.5uL || 40.5uL || 2.0uL || 1.0uL, Xba1 || 1.0uL, Pst1 || 50.0uL
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|}
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|'''5. Vector Dephosphorylate:''' Vector Dephosphorylate the base vector by adding 1uL Antarctic phosphatase and 5uL of Antarctic Phosphatase buffer.
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|'''5. Run a gel:''' Run gel with (1) BV:Tet/p22:RFP and (2) BV:TetRpromoter:LAMBDAcI. Run for 60 minutes @ 80 volts. Results: NO DNA bands found.
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|'''6.  Plasmid Prep the Lambda/LacI:BV cultures:''' Follow same steps as step 2.

Latest revision as of 14:42, 1 August 2008

Back to Notebook Home
Go to Previous Day (July 30)Go to Next Day (August 1)
1. Pick Colonies from BV:Lac/LAMBDA (dual promoter and base vector): Picked colonies and placed BV:dualpromoters in 2mL LB cultures to incubate @ 37C for 8-12 hours. (Also make one 'control' w/ 2mL LB and pipet tip but no colony on it).
2. Plasmid Prep (1) BV:Tet/p22:RFP and (2) BV:Tetpromoter:LAMBDAcIgene: Using QIAmini prep guidelines - prepped 5 samples of BV:TetR/p22:RFP and 5 samples of BV:TetRpromoter:LAMBDAcIgene. Steps: Pour LB culture into tube. Spin tube and remove supernatant. Resuspend pelleted bacterial cells in 250uL Buffer P1 Resuspension buffer. Add 250uL Buffer P2 Lysis buffer and invert 4-6 times. Add 350uL of Buffer N3 Neutralization buffer and invert 4-6 times. Centrifuge for 10 minutes @ 13,000 rpm's. Put supernatants in to spin column, centrifuge for 30-60 seconds (spin column binds to DNA and everything else flows through). Discard flow through. Add 500.0uL of Buffer PB binding buffer, centrifuge for 30-60 seconds, discard flow through. Add 750.0uL Buffer PE wash buffer, centrifuge for 30-60 seconds, discard supernatants and centrifuge for an additional 30-60 seconds. Place spin column in microcentrifuge tube, and elute DNA by adding 50uL Buffer EB elution buffer (10mM Tris-Cl, pH 8.5)let stand for 5 minutes and centrifuge.
NOTE: Last step of Plasmid Prep was performed differently to obtain a higher concentration of DNA. After elution buffer fell through and flow through had DNA in it so repoured that into spin column a second time to spin again for another 60 seconds to extract more DNA.
3. Spectrophotometry: Spec the 10 plasmid prep samples to check for concentration of DNA.
4. Double Digest: Digest Plasmid Prep products. After following procedure below


Parts 10x Buffer BSA H20 DNA RE 1 RE 2 Total
BV + T/P + RFP (1) 5.0uL 0.5uL 2.5uL 40.0uL 1.0uL, Spe1 1.0uL, Pst1 50.0uL
BV + T/P + RFP (2) 5.0uL 0.5uL 2.5uL 40.0uL 1.0uL, Spe1 1.0uL, Pst1 50.0uL
BV + Tetpro. + LAMBDAcI (1) 5.0uL 0.5uL 2.5uL 40.0uL 1.0uL, Spe1 1.0uL, Pst1 50.0uL
BV + Tetpro. + LAMBDAcI (2) 5.0uL 0.5uL 2.5uL 40.0uL 1.0uL, Spe1 1.0uL, Pst1 50.0uL
Terminator 5.0uL 0.5uL 40.5uL 2.0uL 1.0uL, Xba1 1.0uL, Pst1 50.0uL
5. Vector Dephosphorylate: Vector Dephosphorylate the base vector by adding 1uL Antarctic phosphatase and 5uL of Antarctic Phosphatase buffer.
5. Run a gel: Run gel with (1) BV:Tet/p22:RFP and (2) BV:TetRpromoter:LAMBDAcI. Run for 60 minutes @ 80 volts. Results: NO DNA bands found.
6. Plasmid Prep the Lambda/LacI:BV cultures: Follow same steps as step 2.
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