Haynes Lab:Notebook/Engineering PC-TFs/2013/03/25: Difference between revisions
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==Summary== | ==Summary== | ||
* | * Cutting inserts to be placed in mammalian vector | ||
A) Get biobricks and enzymes from freezer. | |||
PstI, SpeI | |||
The following list of parts are: | |||
* Kozak | |||
Follow steps for gel electrophoresis. | |||
* Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray. | |||
* Fill gel flask with up to 60 ml of TA buffer. | |||
* Create 1% gel by putting .6 grams of agarose into flask. | |||
* Microwave agarose solution for 40 seconds | |||
* Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds. | |||
* When flask is taken out of microwave, make sure that the agarose is completed dissolved. | |||
* Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches. | |||
* Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL. | |||
* Pour gel into tray. | |||
* Wash the agarose gel flask. | |||
* Run gel for 45 min at 100 V. Check gel under UV light. | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | '''Reagent''' | |||
| bgcolor=#cfcfcf | '''Volume''' | |||
| rowspan="7" | [[Image:gel3_25_2013.jpg|200px|Stock digest 3/25/2013]] [[Image:gel3_25_2013_cutout.jpg|200px|Stock digest 3/18/2013]] <br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| DNA (plasmid) || 20.0 μL | |||
|- | |||
| SpeI|| 1.0 μL | |||
|- | |||
| PstI|| 1.0 μL | |||
|- | |||
| Green 10x FastDigest buffer || 3.0 μL | |||
|- | |||
| dH<sub>2</sub>O || 5 μL | |||
|- | |||
| || 30 μL --> 37°C/ ~10 min. | |||
|} | |||
{|border="1" cellpadding="5" cellspacing="0" align="left" | |||
|- | |||
! scope="col" style="background:#efefef;" | # | |||
! scope="col" style="background:#efefef;" | Biobrick | |||
! scope="col" style="background:#efefef;" | ng/µL | |||
|- | |||
|1 | |||
|Kozak | |||
|9.47 | |||
|} | |||
Revision as of 18:21, 6 September 2013
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Summary
A) Get biobricks and enzymes from freezer. PstI, SpeI The following list of parts are:
Follow steps for gel electrophoresis.
|