Haynes Lab:Notebook/Engineering PC-TFs/2013/10/11: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(Autocreate 2013/10/11 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs) |
(fix raw html notebook nav) |
||
(3 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span> | |style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></span> | ||
|style="background-color: #800000" align="center"| | |style="background-color: #800000" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==October 11, 2013== | ||
* | '''Restriction Digest''' | ||
''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:abc12.jpg|250px| CMV Stock digest 10/11/2013]] [[Image:whyohwhy.jpg|250px|CMV Stock Digest 10/11/2013]] <br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
| DNA (plasmid) || 20.0 μL | |||
|- | |||
| XbaI|| 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;" | Part | |||
! scope="col" style="background:#efefef;" | ng/µL | |||
|- | |||
|1 | |||
|CMV | |||
|0.851 | |||
|- | |||
|2 | |||
|CMV | |||
|15.535 | |||
|} | |||
Latest revision as of 23:26, 26 September 2017
Main project page Previous entry Next entry | |||||||||||||||||||||||||
October 11, 2013Restriction Digest Gel Electrophoresis
ZymocleanTM Gel DNA Recovery Kit
|