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Team:Evry-Genopole/Notebook/July/5
From 2012.igem.org
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
