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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
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==Summary==
==Summary==
*  
 
 
*'''[[User:Karmella Haynes|---Karmella]] 01:17, 26 November 2012 (EST)''':
 
* Digest the pSB1A2/3 plasmid (from Rene) with XbaI & SpeI to get rid of the insert
 
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
|-valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| rowspan="9" |
|-
| DNA (plasmid) || 20.0
|-
| 10x buffer || 3.0
|-
| XbaI || 1.0
|-
| SpeI || 1.0
|-
| dH<sub>2</sub>O || 5.0
|-
| &nbsp; || 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.
 




Revision as of 18:33, 6 September 2013



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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.



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