Haynes Lab:Notebook/Engineering PC-TFs/2013/09/12: Difference between revisions
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== | ==September 12, 2013== | ||
* | * 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:cmvgel2.jpg|250px| CMV and MV9 Stock digest]] [[Image:cmvgel1_cutout.jpg|250px|CMV and MV9 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. | |||
|} | |||
''Zymoclean<sup>TM</sup> Gel DNA Recovery Kit'' | |||
*Add 3 volumes of ADB Buffer to each volume of gel (600 μL). | |||
*Incubate at 55°C for 10 minutes. | |||
*Centrifuge at ≥ 10,000 x g for 30 seconds. Discard flow-through. | |||
*Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat wash step. | |||
*Place Zymoclean Column into a new 1.5 mL tube. Add 20μL of dH<sub>2</sub>O to elute DNA | |||
{|border="1" cellpadding="5" cellspacing="0" align="left" | |||
|- | |||
! scope="col" style="background:#efefef;" | # | |||
! scope="col" style="background:#efefef;" | Sequence | |||
! scope="col" style="background:#efefef;" | ng/µL | |||
|- | |||
|1 | |||
|CMV | |||
| -1.17 | |||
|- | |||
|2 | |||
|CMV | |||
|2.767 | |||
|- | |||
|3 | |||
|CMV | |||
| -2.116 | |||
|- | |||
|5 | |||
|MV9 | |||
|20.838 | |||
|- | |||
|} | |||
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Revision as of 21:19, 11 October 2013
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September 12, 2013
ZymocleanTM Gel DNA Recovery Kit
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