Minnesota/10 July 2008

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|1. '''Dephosphorylation:''' Continued with double digestion base vector product from 07-09-2008. Used Antartic phosphoylase to dephosphorylate the base vector. Want to dephosphorylate the base vector to prevent it from closing - want it to stay a linear digested product. After dephosphorylation, the base vector was then incubated @ 37C for 30 minutes. Sample was then placed in a heat bath @ 65C for 15 minutes to inactivate enzyme (phosphotase).
|1. '''Dephosphorylation:''' Continued with double digestion base vector product from 07-09-2008. Used Antartic phosphoylase to dephosphorylate the base vector. Want to dephosphorylate the base vector to prevent it from closing - want it to stay a linear digested product. After dephosphorylation, the base vector was then incubated @ 37C for 30 minutes. Sample was then placed in a heat bath @ 65C for 15 minutes to inactivate enzyme (phosphotase).
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| NOTE: Total volume in dephosphorylation = 56.6uL
| NOTE: Total volume in dephosphorylation = 56.6uL
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|[[Image:Dephosphorylation.jpg|thumb|left|600px|Dephosphorylate Base Vector Diagram]]
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|5. '''Ligation''': Follow table below. When ligation is complete, incubate products @16C for 1 hour. Heat inactivate DNA ligase (enzyme) @ 65C for 15 minutes.
|5. '''Ligation''': Follow table below. When ligation is complete, incubate products @16C for 1 hour. Heat inactivate DNA ligase (enzyme) @ 65C for 15 minutes.
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!| Reagent !! Buffer !! H20 !! Insert DNA !! LAMBDAcI DNA !! Dephosphor. Base V. !! T4 DNA Ligase
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|pro-LAMBDAcI-B.V. ||4.0uL || 0||promoter 1.0uL ||6.0uL ||10.0uL ||1.0uL
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|LAMBDAcI-term-B.V. ||4.0uL || 0||terminator 1.0uL ||6.0uL ||10.0uL ||1.0uL
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|Control ||4.0uL || 7.0uL || 0|| 0|| 10.0uL ||1.0uL
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|6. '''Ligation product Transformations''': Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours. Following growth, 200 uL of all four ligation reactions and a control were plated on Kanamycin LB and were incubated at 37 degrees overnight.
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|7. '''Ordered Primers''': Ordered primers for possible use in real-time PCR.
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!| Reagent !!Volume in uL
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!| Primer !!Sequence
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|10x Buffer ||2.0uL
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|F Mcherry for modification|| gaattcgcggccgcttctagatggtgagcaagggcgagg
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|H20 ||20.0uL
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|R Mcherry for modification ||TATAAACGCAGAAAGGCCCACCC
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|Vector ||
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|R LAMcI for realtime PCR || GGTTGTGCTTACCCATCTCTCC
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|DNA ||
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|R p22mnt for realtime PCR || ACTCGCTCTGCTCATCGGCG
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|T4 DNA Ligase || 1.0uL
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|R p22cII for realtime PCR || CAATCTACAGTGGTGTCGTGCC
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|R GFP for realtime PCR || GAATGTTTCCATCTTCTTTAAAATC
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|R mCherry for realtime PCR || GTGATGAACTTCGAGGACGGCG
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|6. '''Ligation product Transformations''': Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours.
 
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Latest revision as of 20:33, 23 July 2008

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1. Dephosphorylation: Continued with double digestion base vector product from 07-09-2008. Used Antartic phosphoylase to dephosphorylate the base vector. Want to dephosphorylate the base vector to prevent it from closing - want it to stay a linear digested product. After dephosphorylation, the base vector was then incubated @ 37C for 30 minutes. Sample was then placed in a heat bath @ 65C for 15 minutes to inactivate enzyme (phosphotase).
Gene 10x Buffer DNA Phosphotase
Base Vector 5.6uL 50.0uL 1.0uL
NOTE: Total volume in dephosphorylation = 56.6uL
Dephosphorylate Base Vector Diagram
2. Run RXN model on Calhoun (super computer).
3. Dilute base vector culture, allow to grow, then miniprep, then streak plates and make base vector glycerol stocks.
4. Autoclave dishes
5. Ligation: Follow table below. When ligation is complete, incubate products @16C for 1 hour. Heat inactivate DNA ligase (enzyme) @ 65C for 15 minutes.
Reagent Buffer H20 Insert DNA LAMBDAcI DNA Dephosphor. Base V. T4 DNA Ligase
pro-LAMBDAcI-B.V. 4.0uL 0promoter 1.0uL 6.0uL 10.0uL 1.0uL
LAMBDAcI-term-B.V. 4.0uL 0terminator 1.0uL 6.0uL 10.0uL 1.0uL
Control 4.0uL 7.0uL 0 0 10.0uL 1.0uL


6. Ligation product Transformations: Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours. Following growth, 200 uL of all four ligation reactions and a control were plated on Kanamycin LB and were incubated at 37 degrees overnight.
7. Ordered Primers: Ordered primers for possible use in real-time PCR.
Primer Sequence
F Mcherry for modification gaattcgcggccgcttctagatggtgagcaagggcgagg
R Mcherry for modification TATAAACGCAGAAAGGCCCACCC
R LAMcI for realtime PCR GGTTGTGCTTACCCATCTCTCC
R p22mnt for realtime PCR ACTCGCTCTGCTCATCGGCG
R p22cII for realtime PCR CAATCTACAGTGGTGTCGTGCC
R GFP for realtime PCR GAATGTTTCCATCTTCTTTAAAATC
R mCherry for realtime PCR GTGATGAACTTCGAGGACGGCG
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