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(7/1/14 Gel Electrophoresis)
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
 
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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|style="background-color: #F2F2F2" 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]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
 
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* Pictures were taken of both gels and data recorded.  
 
* Pictures were taken of both gels and data recorded.  
  
 
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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|- valign="top"
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| bgcolor=#cfcfcf | Uncut Plasmid Check
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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| 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>
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| 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]
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|-
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||| DNA(plasmid) || 5.0 μL
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|-
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||| 10X buffer || 2.0
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|-
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||| dH<sub>2</sub>O || 13.0
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|-
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||| &nbsp; || 20.0 μL --> 37°C/ ~40 min.
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|}
  
  
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
|- valign="top"
 
|- valign="top"
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| bgcolor=#cfcfcf | Insert Size Check
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Volume  
 
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
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| 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]
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| 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]
 
|-
 
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| DNA(plasmid) || 2.0 μL
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||| DNA(plasmid) || 5.0 μL
 
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| 10X buffer || 1.5
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||| 10X buffer || 2.0
 
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| EcoRI || 1.0
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||| EcoRI || 1.0
 
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| PstI || 1.0
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||| PstI || 1.0
 
|-
 
|-
| dH<sub>2</sub>O || 9.5
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||| dH<sub>2</sub>O || 11.0
 
|-
 
|-
| &nbsp; || 15 μL --> 37°C/ ~40 min.
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||| &nbsp; || 20.0 μL --> 37°C/ ~40 min.
 
|}
 
|}
  

Latest revision as of 23:04, 26 September 2017

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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) 5.0 μL
10X buffer 2.0
dH2O 13.0
  20.0 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) 5.0 μL
10X buffer 2.0
EcoRI 1.0
PstI 1.0
dH2O 11.0
  20.0 μL --> 37°C/ ~40 min.