Team:Washington/Protocols/test

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[[File:Allkane_groups.png]]
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<a href="http://www4.clustrmaps.com/user/a18e3316"><img src="http://www4.clustrmaps.com/stats/maps-no_clusters/2011.igem.org-Team-Washington--thumb.jpg" alt="Locations of visitors to this page" />
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'''Restriction Digest''' (10 uL DNA)
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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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Latest revision as of 23:09, 28 September 2011

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