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| - | {{Template:Team:Washington/Templates/Top}}
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| - | {{abcde}}
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| - | INSERT INFO HERE... LOOK AT OTHER PAGES FOR EXAMPLES OF FORMATING AND INSERTING PICTURES. CLICK THE EDIT BUTTON ON THE UPPER LEFT SIDE OF THE PAGE AFTER YOU HAVE SIGNED IN.
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| - | [https://2011.igem.org/Team:Washington/Alkanes/Protocols/Example_1 Example 1]
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| - | [https://2011.igem.org/Team:Washington/Alkanes/Protocols/Example_2 Example 2]
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| - | [https://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagensis]
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| - | [http://www.bio.davidson.edu/Courses/molbio/kunkel/kunkel.html Overview of how Kunkel Mutagensis works]
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| - | [https://2011.igem.org/Team:Washington/Protocols/plate_expression 96 Well Plate Protein Expression]
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| - | [https://2011.igem.org/Team:Washington/alkanebiosynthesis Alkane Biosynthesis media and extraction]
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| - | [https://2011.igem.org/Team:Washington/Protocols/expression_purification Standard 1L Expression Purification]
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| - | [https://2011.igem.org/Team:Washington/Protocols/gene_assembly Gene Assembly With Oligos]
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| - | [https://2011.igem.org/Team:Washington/Protocols/test]
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| - | =Protocol Page=
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| - | '''restriction digest'''
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| - | 10 uL DNA
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| - | 5uL buffer ( 2 for most, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp)
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| - | .5 uL BSA
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| - | 1uL enzyme 1
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| - | 1uL enzyme 2
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| - | water to 50 uL(32.5 uL, add first)
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| - | '''oligo assembly by PCR'''
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| - | resuspend oligos with water, amount of water= concentration(in nm)*10 in uL
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| - | make oligo mix with 5uL of each primer
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| - | PCR reaction:
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| - | 1uL phusion
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| - | .5uL oligo mix
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| - | 1uL first oligo
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| - | 1uL last oligo
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| - | 5uL buffer
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| - | 1uL dnTP
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| - | dH20 to 50uL
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| - | '''Ligation'''
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| - | 7uL insert
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| - | 1uL vector
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| - | 1uL T4 ligase buffer
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| - | 1uL T4 ligase
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| - | incubate at <s>37C</s> no. **Note that NEB website recommends 16C - room temp for ligations, we do ours at RT and it works well. 1 hour.
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| - | '''Colony PCR with Green tag'''
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| - | Master mix(7ul):
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| - | 1ul 10uM forword primer
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| - | 1ul 10uM reverse Primer
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| - | 5ul 2x Green tag
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| - | Cell water(3ul): Pick one colony from the plate and mix with 10ul of ddH2O to make 10ul of cell water
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| - | Reaction = Master mix(7ul) + Cell water(3ul) = 10ul per tube
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| - | Use program "Colony" & change the extention time (1min per kb)
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| - | '''Heat Shock Transformation'''
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| - | 2 ul ligation
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| - | 20 ul cells
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| - | Ice 20 minutes
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| - | Heat shock at 42C for 1 minute
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| - | Ice 2 minutes
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| - | Prepare 200 ul of TB (no anti) and transformed cells in culture tube
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| - | Incubate at 37C for 1 hour
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| - | Plate cells
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