From 2012.igem.org

Revision as of 15:43, 12 July 2012

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)

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

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

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