Difference between revisions of "Haynes Lab:Notebook/Engineering PC-TFs/2012/11/26"

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(Autocreate 2012/11/26 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
 
(Summary)
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==Summary==
 
==Summary==
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*'''[[User:Karmella Haynes|---Karmella]] 01:17, 26 November 2012 (EST)''':
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* Digest the pSB1A2/3 plasmid (from Rene) with XbaI & SpeI to get rid of the insert
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
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|-valign="top"
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| <u>Reagent</u> || <u>Volume</u> || &nbsp;
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| rowspan="9" |
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|-
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| DNA (plasmid) || 20.0
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|-
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| 10x buffer || 3.0
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|-
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| XbaI || 1.0
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|-
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| SpeI || 1.0
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|-
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| dH<sub>2</sub>O || 5.0
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|-
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| &nbsp; || 30 μL --> 37°C/ ~10 min.
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|}
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* Run entire 30 μL on a 1% gel (use big tooth comb) next to a ladder
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* You should see two bands. The larger band (2000 bp) is the backbone.
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* Cut out and gel purify this fragment (ignore the shorter fragment).
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* Notify Dr. Haynes when you are done. The next step is Gibson Assembly and transformation.
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Revision as of 22:17, 25 November 2012

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Summary

  • Digest the pSB1A2/3 plasmid (from Rene) with XbaI & SpeI to get rid of the insert
Reagent Volume  
DNA (plasmid) 20.0
10x buffer 3.0
XbaI 1.0
SpeI 1.0
dH2O 5.0
  30 μL --> 37°C/ ~10 min.
  • Run entire 30 μL on a 1% gel (use big tooth comb) next to a ladder
  • You should see two bands. The larger band (2000 bp) is the backbone.
  • Cut out and gel purify this fragment (ignore the shorter fragment).
  • Notify Dr. Haynes when you are done. The next step is Gibson Assembly and transformation.