Team:Evry/Notebook/July/5

From 2012.igem.org

(Difference between revisions)
Line 6: Line 6:
<FONT SIZE=3>'''Gel Extraction'''</FONT> <br/>
<FONT SIZE=3>'''Gel Extraction'''</FONT> <br/>
 +
<br/>
1) Excise the DNA fragment from agarose gel<br/>
1) Excise the DNA fragment from agarose gel<br/>
2) Weight the gel slice<br/>
2) Weight the gel slice<br/>
Line 11: Line 12:
<TABLE BORDER="1">
<TABLE BORDER="1">
   <TR>
   <TR>
-
     <TH> Type </TH>
+
     <TH> <center> Type </center></TH>
-
     <TH> Gel weight (g) </TH>
+
     <TH> <center>Gel weight (g) </center></TH>
-
     <TH> V QG (uL) </TH>
+
     <TH> <center>V QG (uL) </center></TH>
-
     <TH> V iso (uL) </TH>
+
     <TH> <center>V iso (uL) </center></TH>
   </TR>
   </TR>
   <TR>
   <TR>
     <TH> pCS2 </TH>
     <TH> pCS2 </TH>
-
     <TD> 0,13 </TD>
+
     <TD> <center>0,13 </center></TD>
-
     <TD> 390 </TD>
+
     <TD> <center>390 </center></TD>
-
     <TD> 130 </TD>
+
     <TD> <center>130 </center></TD>
   </TR>
   </TR>
   <TR>
   <TR>
     <TH> GFP </TH>
     <TH> GFP </TH>
-
     <TD> 0,10 </TD>
+
     <TD> <center>0,10 </center></TD>
-
     <TD> 300 </TD>
+
     <TD> <center>300 </center></TD>
-
     <TD> 100 </TD>
+
     <TD> <center>100 </center></TD>
   </TR>
   </TR>
   <TR>
   <TR>
     <TH> YFP </TH>
     <TH> YFP </TH>
-
     <TD> 0,08 </TD>
+
     <TD> <center>0,08 </center></TD>
-
     <TD> 240 </TD>
+
     <TD> <center>240 </center></TD>
-
     <TD> 80 </TD>
+
     <TD> <center>80 </center></TD>
   </TR>
   </TR>
   <TR>
   <TR>
     <TH> CFP </TH>
     <TH> CFP </TH>
-
     <TD> 0,10 </TD>
+
     <TD> <center>0,10 </center></TD>
-
     <TD> 300 </TD>
+
     <TD> <center>300 </center></TD>
-
     <TD> 100 </TD>
+
     <TD> <center>100 </center></TD>
   </TR>
   </TR>
</TABLE>
</TABLE>
-
 
+
<br/>
4) Incubate at 50 degrees Celsius for 10 min to dissolve the gel<br/>
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/>
5) Add 1 gel volume of isopropanol to the sample and mix<br/>
-
6) Apply the sample to the QIAquick column to bond DNA
+
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/>
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/>
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/>
9) Centrifuge at 13 000 rpm for 1 min and discard flow-through<br/>
-
10) To wash, add 750 uL of Buffer PE  
+
10) To wash, add 750 uL of Buffer PE <br/>
11) Centrifuge at 13 000 rpm for 1 min and discard flow-through<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/>
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
+
13) Add 50 uL of Buffer EB to elute DNA<br/>
-
14) Check DNA concentration with Nanodrop
+
14) Check DNA concentration with Nanodrop<br/>
<br/>
<br/>
<FONT SIZE=3>'''DNA Concentrations'''</FONT> <br/>
<FONT SIZE=3>'''DNA Concentrations'''</FONT> <br/>
 +
<br/>
unit= ng/uL<br/>
unit= ng/uL<br/>
CFP : 4,7 ng/uL<br/>
CFP : 4,7 ng/uL<br/>
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pCS2+ : 9,4 ng/uL<br/>
pCS2+ : 9,4 ng/uL<br/>
<br/>
<br/>
-
Concentration are really low => Need to optimize the protocol and make a new gel migration.
+
 
 +
<TABLE BORDER="1">
 +
  <CAPTION> DNA Concentration </CAPTION>
 +
  <TR>
 +
    <TH> <center> Type </center></TH>
 +
    <TH> <center> Concentration (ng.uL-1) </center></TH>
 +
  </TR>
 +
  <TR>
 +
    <TH> pCS2 </TH>
 +
    <TD> <center>9,4 </center></TD>
 +
  </TR>
 +
  <TR>
 +
    <TH> GFP </TH>
 +
    <TD> <center>3,1 </center></TD>
 +
  </TR>
 +
  <TR>
 +
    <TH> YFP </TH>
 +
    <TD> <center>4,7 </center></TD>
 +
  </TR>
 +
  <TR>
 +
    <TH> CFP </TH>
 +
    <TD> <center>4,7 </center></TD>
 +
  </TR>
 +
</TABLE>
 +
 
 +
<br/>
 +
Concentration are really low => Need to optimize the protocol and make a new gel migration.<br/>
<br/>
<br/>
<FONT SIZE=3>'''Gel Migration'''</FONT> <br/>
<FONT SIZE=3>'''Gel Migration'''</FONT> <br/>
-
Gel at 0,08% <br/>
 
<br/>
<br/>
-
V tae = 30 mL
+
Gel at 0,08% <br/>
-
m aga = 0,24 g  
+
V tae = 30 mL<br/>
-
V bet = 3 uL
+
m aga = 0,24 g <br/>
 +
V bet = 3 uL<br/>
<br/>
<br/>

Revision as of 15:32, 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

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

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