IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/01

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(7/1/14 Gel Electrophoresis)
(7/1/14 Gel Electrophoresis)
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* Pictures were taken of both gels and data recorded.  
* 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) || 2.0 μL
 +
|-
 +
||| 10X buffer || 1.5
 +
|-
 +
||| EcoRI || 1.0
 +
|-
 +
||| PstI || 1.0
 +
|-
 +
||| dH<sub>2</sub>O || 9.5
 +
|-
 +
||| &nbsp; || 15 μL --> 37°C/ ~40 min.
 +
|}
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
|- valign="top"
 +
| bgcolor=#cfcfcf | Insert Size Check
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| 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= 2080<br>
+
| 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_concentrationgel.JPG|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
+
| 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) || 2.0 μL
+
||| DNA(plasmid) || 2.0 μL
|-
|-
-
| 10X buffer || 1.5
+
||| 10X buffer || 1.5
|-
|-
-
| EcoRI || 1.0
+
||| EcoRI || 1.0
|-
|-
-
| PstI || 1.0
+
||| PstI || 1.0
|-
|-
-
| dH<sub>2</sub>O || 9.5
+
||| dH<sub>2</sub>O || 9.5
|-
|-
-
| &nbsp; || 15 μL --> 37°C/ ~40 min.
+
||| &nbsp; || 15 μL --> 37°C/ ~40 min.
|}
|}

Revision as of 13:31, 15 July 2014

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7/1/14 Gel Electrophoresis

  • 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.
Uncut Plasmid Check Reagent Volume Expected:
1. TesA = 2721
2. mCherry = size
3. TetR = 2124
4. Lac = 2125
5. RBS= 2063
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~40 min.


Insert Size Check Reagent Volume Expected:
All Backbones = 2070 1. TesA = 651
2. mCherry = size
3. TetR = 54
4. Lac = 55
5. RBS= 13
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~40 min.


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