IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/01: Difference between revisions
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== | ==7/1/14 Gel Electrophoresis== | ||
* Insert | * '''Gel Electrophoresis of Plasmids''' | ||
* Made TAE buffer with 1 L of distilled water and 20mL of 50X stock of TAE. Also prepared the gel with agarose and water mixture and let solidify. | |||
* Made plasmids ready by using 3μL of DNA and 3μL of loading dye. 5 tubes of this were made as well as 1 tube of ladder 3μL. | |||
* Insert ladder as well as DNA plasmids in wells and let sit for around 45-50 minutes. | |||
* This was to determine concentration of plasmids | |||
*Next, the sizes of the plasmids were tested to see how accurate they are compared to the literature value | |||
*Used the plasmids and cut them with restriction digests | |||
*2μL of DNA, 1μL of Buffer, 5μL of water, 2μL of, 1μL of E and 1μ of P restriction digests. | |||
* Heated in a 37°C water bath for around 40 minutes | |||
* Plasmids that have been cut were taken out and loading dye was input--2μL | |||
*This along with the ladder was filled into the wells and the machine was run for around 35 minutes. The voltage was increased from 80 V to 100 V | |||
* Pictures were taken of both gels and data recorded. | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Uncut Plasmid Check | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br> 1. TesA = 2721<br>2. mCherry = size<br> 3. TetR = 2124<br> 4. Lac = 2125<br> 5. RBS= 2063<br> | |||
| rowspan="7" | [[Image:Asuigem2014_07012014_concentrationgel.JPG|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
||| DNA(plasmid) || 5.0 μL | |||
|- | |||
||| 10X buffer || 2.0 | |||
|- | |||
||| dH<sub>2</sub>O || 13.0 | |||
|- | |||
||| || 20.0 μL --> 37°C/ ~40 min. | |||
|} | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Insert Size Check | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br> All Backbones = 2070 1. TesA = 651<br>2. mCherry = size<br> 3. TetR = 54<br> 4. Lac = 55<br> 5. RBS= 13<br> | |||
| rowspan="7" | [[Image:Asuigem2014_07012014_insertsizetest.JPG|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |||
||| DNA(plasmid) || 5.0 μL | |||
|- | |||
||| 10X buffer || 2.0 | |||
|- | |||
||| EcoRI || 1.0 | |||
|- | |||
||| PstI || 1.0 | |||
|- | |||
||| dH<sub>2</sub>O || 11.0 | |||
|- | |||
||| || 20.0 μL --> 37°C/ ~40 min. | |||
|} | |||
Revision as of 10:41, 15 July 2014
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7/1/14 Gel Electrophoresis
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