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| - | <span style="font-family: Century gothic; font-size: 10pt"> | + | ROkTtl <a href="http://nvclxegnjthc.com/">nvclxegnjthc</a>, [url=http://weezbkxumhlh.com/]weezbkxumhlh[/url], [link=http://ppdjqipvcajn.com/]ppdjqipvcajn[/link], http://ewkvqyyyiere.com/ |
| - | <div style="text-align: right;"> [[Team:UC_Berkeley/Notebook/Sherine_Cheung|Back to Sherine's Notebook]] </div> | + | |
| - | <div style="text-align: right;"> [[Team:UC_Berkeley/Notebook| To All Notebooks]] </div>
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| - | ==Newest EcoRI/BamHI Transfer method (7/31/08)==
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| - | Digestion:
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| - | *0.5ul plasmid in entry vector
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| - | *0.5ul EcoRI
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| - | *0.5ul BamHI
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| - | *1ul NEB2 + ATP buffer
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| - | *7.5ul of water
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| - | *incubate the digestion for 1hr. in the thermocycler, and 20 min for heat kill.
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| - | No purification step.
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| - | Ligation:
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| - | *1ul of dilute assembly vector (or 0.5ul if undiluted)
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| - | *0.5ul ligase
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| - | Transform. (same as usual)
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| - | == Transfers from Miniprepped DNA ==
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| - | (already in correct vectors - transfers into L/R cells)
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| - | In 0.5 ml tube, combine:
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| - | *1ul plasmid dna
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| - | *30ul of cells (L/R) + 30 KCM (no water)
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| - |
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| - | Take the reaction, and:
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| - | *sit in ice for 10 min
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| - | *heat shock for 90 sec
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| - | *sit another 2 min ice
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| - | Then, incubate your tubes for 5 minutes (No rescuing is necessary)
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| - | Next, prepare plates (double antibiotics) and tubes with media (LB + antibiotics)
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| - | *Take 15ul of rxn. Spot it onto a plate (no spreading required)
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| - | *Take the remaining 15ul into a pipette tip. Drop the entire tip into your tube of media.
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| - | *Let plates and tubes sit overnight.
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| - | *Miniprep the next day.
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| - | ==1-2-3 Assembly==
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| - | Set up the digestion:
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| - | *1uL lefty plasmid(methylated)
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| - | *1uL righty plasmid (methylated)
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| - | *1uL NEB2+ATP
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| - | *0.3 uL XhoI
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| - | *0.3 uL BglII
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| - | *0.3 uL BamHI
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| - | *6.1uL Water
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| - | Heat kill at 65 for 20 min
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| - | Add 0.3uL Ligase
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| - | Transform, rescue, plate on dual antibiotic plates
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Latest revision as of 15:41, 28 March 2016
ROkTtl <a href="http://nvclxegnjthc.com/">nvclxegnjthc</a>, [url=http://weezbkxumhlh.com/]weezbkxumhlh[/url], [link=http://ppdjqipvcajn.com/]ppdjqipvcajn[/link], http://ewkvqyyyiere.com/
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