From 2011.igem.org
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| | '''[[image:Allkane_groups.png]][[image:v019_tif.jpg|46x30px]]''' | | '''[[image:Allkane_groups.png]][[image:v019_tif.jpg|46x30px]]''' |
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| | 5uL buffer ( 2 for most, check NEB) | | 5uL buffer ( 2 for most, check NEB) |
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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: 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 37C 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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Revision as of 05:55, 12 September 2011
{Template:Css}
46x30px
Restriction Digest (10 uL DNA)
5uL buffer ( 2 for most, check NEB)
"
