Qiagen MinElute Gel Extraction
Excise the DNA fragment from the agarose gel with a clean , sharp scalpel .
Weigh the gel slice in a colorless tube .
Add 3 volumes Buffer QG to 1 volume gel ( 100 mg ~ 100 μl ) .
For > 2 % agarose gels , add 6 volumes Buffer QG .
Incubate at 50°C for 10 min ( or until the gel slice has completely dissolved ) .
Vortex the tube every 2–3 min to help dissolve gel .
After the gel slice has dissolved completely , check that the color of the mixture is yellow ( similar to Buffer QG without dissolved agarose ) .
If the color of the mixture is orange or violet , add 10 μl 3 M sodium acetate , pH 5.0 , and mix .
The color of the mixture will turn yellow .
Add 1 gel volume of isopropanol to the sample and mix .
Place a QIAquick spin column into a provided 2 ml collection tube .
To bind DNA , apply the sample to the QIAquick column and centrifuge for 1 min .
Discard flow - through and place the QIAquick column back into the same tube .
For sample volumes of > 800 μl , load and spin / apply vacuum again .
If the DNA will subsequently be used for sequencing , in vitro transcription , or microinjection , add 0.5 ml Buffer QG to the QIAquick column and centrifuge for 1 min .
Discard flow - through and place the QIAquick column back into the same tube .
To wash , add 0.75 ml Buffer PE to QIAquick column and centrifuge for 1 min .
Note : If the DNA will be used for salt - sensitive applications ( e . g . , sequencing , blunt - ended ligation ) , let the column stand 2–5 min after addition of Buffer PE .
Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min at 17,900 x g ( 13,000 rpm ) to remove residual wash buffer .
Place QIAquick column into a clean 1.5 ml microcentrifuge tube .
To elute DNA , add 10 µl of Buffer EB ( 10 mM Tris·Cl , pH 8.5 ) or water to the center of the membrane , let the column stand for 1 min , and then centrifuge for 1 min .
IMPORTANT : Ensure that the elution buffer is dispensed directly onto the center of the membrane for complete elution of bound DNA .
The average eluate volume is 9 µl from 10 µl elution buffer volume . Elution efficiency is dependent on pH .
The maximum elution efficiency is achieved between pH 7.0 and 8.5 .
When using water , make sure that the pH value is within this range , and store DNA at –20°C as DNA may degrade in the absence of a buffering agent .
The purified DNA can also be eluted in TE buffer ( 10 mM Tris·Cl , 1 mM EDTA , pH 8.0 ) , but the EDTA may inhibit subsequent enzymatic reactions .
If the purified DNA is to be analyzed on a gel , add 1 volume of Loading Dye to 5 volumes of purified DNA .
Mix the solution by pipetting up and down before loading the gel .
Loading dye contains 3 marker dyes ( bromophenol blue , xylene cyanol , and orange G ) that facilitate estimation of DNA migration distance and optimization of agarose gel run time .
Refer to Table 3 ( page 15 ) to identify the dyes according to migration distance and agarose gel percentage and type .
