Team:Evry/Notebook/July/5

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{{:Team:Evry/template_v1}}
<html>
<html>
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<center><h1>Promoters & Reporters workgroup</h1></center>
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</html>
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<FONT SIZE=3>'''Gel Extraction'''</FONT> <br/>
 +
<br/>
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1) Excise the DNA fragment from agarose gel<br/>
 +
2) Weight the gel slice<br/>
 +
3) Add 3 volumes of Buffer QG to 1 volume of gel (100 mg = 100 uL)<br/>
 +
<TABLE BORDER="1">
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  <TR>
 +
    <TH> <center> Type </center></TH>
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    <TH> <center>Gel weight (g) </center></TH>
 +
    <TH> <center>V QG (uL) </center></TH>
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    <TH> <center>V iso (uL) </center></TH>
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  </TR>
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  <TR>
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    <TH> pCS2 </TH>
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    <TD> <center>0,13 </center></TD>
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    <TD> <center>390 </center></TD>
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    <TD> <center>130 </center></TD>
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  </TR>
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  <TR>
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    <TH> GFP </TH>
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    <TD> <center>0,10 </center></TD>
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    <TD> <center>300 </center></TD>
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    <TD> <center>100 </center></TD>
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  </TR>
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  <TR>
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    <TH> YFP </TH>
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    <TD> <center>0,08 </center></TD>
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    <TD> <center>240 </center></TD>
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    <TD> <center>80 </center></TD>
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  </TR>
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  <TR>
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    <TH> CFP </TH>
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    <TD> <center>0,10 </center></TD>
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    <TD> <center>300 </center></TD>
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    <TD> <center>100 </center></TD>
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  </TR>
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</TABLE>
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<br/>
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4) Incubate at 50 degrees Celsius for 10 min to dissolve the gel<br/>
 +
5) Add 1 gel volume of isopropanol to the sample and mix<br/>
 +
6) Apply the sample to the QIAquick column to bond DNA<br/>
 +
7) Centrifuge at 13 000 rpm for 1 min and discard flow-through<br/>
 +
8) Add 500 uL of Buffer QG to the column<br/>
 +
9) Centrifuge at 13 000 rpm for 1 min and discard flow-through<br/>
 +
10) To wash, add 750 uL of Buffer PE <br/>
 +
11) Centrifuge at 13 000 rpm for 1 min and discard flow-through<br/>
 +
12) Centrifuge the column again at 13 000 rpm for 1 min and place the column in a clean 1.5 mL tube<br/>
 +
13) Add 50 uL of Buffer EB to elute DNA<br/>
 +
14) Check DNA concentration with Nanodrop<br/>
 +
<br/>
 +
 +
<TABLE BORDER="1">
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  <CAPTION> DNA Concentration </CAPTION>
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  <TR>
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    <TH> <center> Type </center></TH>
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    <TH> <center> Concentration (ng.uL-1) </center></TH>
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  </TR>
 +
  <TR>
 +
    <TH> pCS2 </TH>
 +
    <TD> <center>9,4 </center></TD>
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  </TR>
 +
  <TR>
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    <TH> GFP </TH>
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    <TD> <center>3,1 </center></TD>
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  </TR>
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  <TR>
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    <TH> YFP </TH>
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    <TD> <center>4,7 </center></TD>
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  </TR>
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  <TR>
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    <TH> CFP </TH>
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    <TD> <center>4,7 </center></TD>
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  </TR>
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</TABLE>
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<br/>
 +
Concentration are really low => Need to optimize the protocol and make a new gel migration.<br/>
 +
<br/>
 +
<FONT SIZE=3>'''Gel Migration'''</FONT> <br/>
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<br/>
 +
Gel at 0,08% <br/>
 +
V tae = 30 mL<br/>
 +
m aga = 0,24 g <br/>
 +
V bet = 3 uL<br/>
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<br/>
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No Band for RFP<br/>
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<br/>
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<FONT SIZE=3>'''Gel Extraction 2'''</FONT> <br/>
 +
<br/>
 +
<TABLE BORDER="1">
 +
  <TR>
 +
    <TH> <center> Type </center></TH>
 +
    <TH> <center>Gel weight (g) </center></TH>
 +
    <TH> <center>V QG (uL) </center></TH>
 +
    <TH> <center>V iso (uL) </center></TH>
 +
  </TR>
 +
  <TR>
 +
    <TH> pCS2 </TH>
 +
    <TD> <center>0,084 </center></TD>
 +
    <TD> <center>252 </center></TD>
 +
    <TD> <center>84 </center></TD>
 +
  </TR>
 +
  <TR>
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    <TH> GFP </TH>
 +
    <TD> <center>0,040 </center></TD>
 +
    <TD> <center>120</center></TD>
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    <TD> <center>40</center></TD>
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  </TR>
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  <TR>
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    <TH> YFP </TH>
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    <TD> <center>0,023 </center></TD>
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    <TD> <center>69</center></TD>
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    <TD> <center>23</center></TD>
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  </TR>
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  <TR>
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    <TH> CFP </TH>
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    <TD> <center>0,050 </center></TD>
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    <TD> <center>150</center></TD>
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    <TD> <center>50</center></TD>
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  </TR>
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</TABLE>
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<br/>
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<html>
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<FONT SIZE=3> Protocole modification </FONT> </br>
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Elution in two times 25 uL with water RNAse free instead of EB buffer.</br>
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Latest revision as of 15:45, 12 July 2012

Promoters & Reporters workgroup

Gel Extraction

1) Excise the DNA fragment from agarose gel
2) Weight the gel slice
3) Add 3 volumes of Buffer QG to 1 volume of gel (100 mg = 100 uL)

Type
Gel weight (g)
V QG (uL)
V iso (uL)
pCS2
0,13
390
130
GFP
0,10
300
100
YFP
0,08
240
80
CFP
0,10
300
100


4) Incubate at 50 degrees Celsius for 10 min to dissolve the gel
5) Add 1 gel volume of isopropanol to the sample and mix
6) Apply the sample to the QIAquick column to bond DNA
7) Centrifuge at 13 000 rpm for 1 min and discard flow-through
8) Add 500 uL of Buffer QG to the column
9) Centrifuge at 13 000 rpm for 1 min and discard flow-through
10) To wash, add 750 uL of Buffer PE
11) Centrifuge at 13 000 rpm for 1 min and discard flow-through
12) Centrifuge the column again at 13 000 rpm for 1 min and place the column in a clean 1.5 mL tube
13) Add 50 uL of Buffer EB to elute DNA
14) Check DNA concentration with Nanodrop

DNA Concentration
Type
Concentration (ng.uL-1)
pCS2
9,4
GFP
3,1
YFP
4,7
CFP
4,7


Concentration are really low => Need to optimize the protocol and make a new gel migration.

Gel Migration

Gel at 0,08%
V tae = 30 mL
m aga = 0,24 g
V bet = 3 uL

No Band for RFP

Gel Extraction 2

Type
Gel weight (g)
V QG (uL)
V iso (uL)
pCS2
0,084
252
84
GFP
0,040
120
40
YFP
0,023
69
23
CFP
0,050
150
50


Protocole modification
Elution in two times 25 uL with water RNAse free instead of EB buffer.

Retrieved from "http://2012.igem.org/Team:Evry/Notebook/July/5"