Team:Evry/Notebook/July/5
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+ | <center><h1>Promoters & Reporters workgroup</h1></center> | ||
+ | </html> | ||
+ | |||
+ | <FONT SIZE=3>'''Gel Extraction'''</FONT> <br/> | ||
+ | 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"> | ||
+ | <TR> | ||
+ | <TH> Type </TH> | ||
+ | <TH> Gel weight (g) </TH> | ||
+ | <TH> V QG (uL) </TH> | ||
+ | <TH> V iso (uL) </TH> | ||
+ | </TR> | ||
+ | <TR> | ||
+ | <TH> pCS2 </TH> | ||
+ | <TD> 0,13 </TD> | ||
+ | <TD> 390 </TD> | ||
+ | <TD> 130 </TD> | ||
+ | </TR> | ||
+ | <TR> | ||
+ | <TH> GFP </TH> | ||
+ | <TD> 0,10 </TD> | ||
+ | <TD> 300 </TD> | ||
+ | <TD> 100 </TD> | ||
+ | </TR> | ||
+ | <TR> | ||
+ | <TH> YFP </TH> | ||
+ | <TD> 0,08 </TD> | ||
+ | <TD> 240 </TD> | ||
+ | <TD> 80 </TD> | ||
+ | </TR> | ||
+ | <TR> | ||
+ | <TH> CFP </TH> | ||
+ | <TD> 0,10 </TD> | ||
+ | <TD> 300 </TD> | ||
+ | <TD> 100 </TD> | ||
+ | </TR> | ||
+ | </TABLE> | ||
+ | |||
+ | 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 | ||
+ | 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 | ||
+ | 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 | ||
+ | 14) Check DNA concentration with Nanodrop | ||
+ | <br/> | ||
+ | <FONT SIZE=3>'''DNA Concentrations'''</FONT> <br/> | ||
+ | unit= ng/uL<br/> | ||
+ | CFP : 4,7 ng/uL<br/> | ||
+ | GFP : 3,1 ng/uL<br/> | ||
+ | YFP : 4,7 ng/uL<br/> | ||
+ | pCS2+ : 9,4 ng/uL<br/> | ||
+ | <br/> | ||
+ | Concentration are really low => Need to optimize the protocol and make a new gel migration. | ||
+ | <br/> | ||
+ | <FONT SIZE=3>'''Gel Migration'''</FONT> <br/> | ||
+ | Gel at 0,08% <br/> | ||
+ | <br/> | ||
+ | V tae = 30 mL | ||
+ | m aga = 0,24 g | ||
+ | V bet = 3 uL | ||
+ | <br/> | ||
+ | <FONT SIZE=3>'''Gel Extraction'''</FONT> <br/> | ||
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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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