Haynes Lab:Notebook/Engineering PC-TFs/2012/11/26: Difference between revisions
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(Autocreate 2012/11/26 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs) |
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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> || | |||
| rowspan="9" | | |||
|- | |||
| DNA (plasmid) || 20.0 | |||
|- | |||
| 10x buffer || 3.0 | |||
|- | |||
| XbaI || 1.0 | |||
|- | |||
| SpeI || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 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. | |||
Revision as of 23:17, 25 November 2012
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Summary
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