Team:Evry/Notebook/July/5

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<center><h1>Promoters & Reporters workgroup</h1></center>
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<FONT SIZE=3>'''Gel Extraction'''</FONT> <br/>
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1) Excise the DNA fragment from agarose gel<br/>
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2) Weight the gel slice<br/>
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3) Add 3 volumes of Buffer QG to 1 volume of gel (100 mg = 100 uL)<br/>
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<TABLE BORDER="1">
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  <TR>
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    <TH> Type </TH>
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    <TH> Gel weight (g) </TH>
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    <TH> V QG (uL) </TH>
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    <TH> V iso (uL) </TH>
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  </TR>
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  <TR>
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    <TH> pCS2 </TH>
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    <TD> 0,13 </TD>
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    <TD> 390 </TD>
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    <TD> 130 </TD>
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  </TR>
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  <TR>
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    <TH> GFP </TH>
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    <TD> 0,10 </TD>
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    <TD> 300 </TD>
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    <TD> 100 </TD>
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  </TR>
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  <TR>
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    <TH> YFP </TH>
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    <TD> 0,08 </TD>
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    <TD> 240 </TD>
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    <TD> 80 </TD>
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  </TR>
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  <TR>
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    <TH> CFP </TH>
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    <TD> 0,10 </TD>
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    <TD> 300 </TD>
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    <TD> 100 </TD>
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  </TR>
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</TABLE>
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4) Incubate at 50 degrees Celsius for 10 min to dissolve the gel<br/>
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5) Add 1 gel volume of isopropanol to the sample and mix<br/>
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6) Apply the sample to the QIAquick column to bond DNA
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7) Centrifuge at 13 000 rpm for 1 min and discard flow-through<br/>
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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
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11) Centrifuge at 13 000 rpm for 1 min and discard flow-through<br/>
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12) Centrifuge the column again at 13 000 rpm for 1 min and place the column in a clean 1.5 mL tube<br/>
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13) Add 50 uL of Buffer EB to elute DNA
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14) Check DNA concentration with Nanodrop
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<br/>
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<FONT SIZE=3>'''DNA Concentrations'''</FONT> <br/>
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unit= ng/uL<br/>
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CFP : 4,7 ng/uL<br/>
 +
GFP : 3,1 ng/uL<br/>
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YFP : 4,7 ng/uL<br/>
 +
pCS2+ : 9,4 ng/uL<br/>
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<br/>
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Concentration are really low => Need to optimize the protocol and make a new gel migration.
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<br/>
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<FONT SIZE=3>'''Gel Migration'''</FONT> <br/>
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Gel at 0,08% <br/>
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<br/>
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V tae = 30 mL
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m aga = 0,24 g
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V bet = 3 uL
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<br/>
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<FONT SIZE=3>'''Gel Extraction'''</FONT> <br/>
<a href="https://2012.igem.org/Team:Evry/Notebook/July/6"><img  src="https://static.igem.org/mediawiki/2012/e/e3/Next_day_arrow.png"></a>
<a href="https://2012.igem.org/Team:Evry/Notebook/July/6"><img  src="https://static.igem.org/mediawiki/2012/e/e3/Next_day_arrow.png"></a>
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Revision as of 14:43, 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 Concentrations
unit= ng/uL
CFP : 4,7 ng/uL
GFP : 3,1 ng/uL
YFP : 4,7 ng/uL
pCS2+ : 9,4 ng/uL

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
Gel Extraction

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