Qiagen QIAquick PCR Purification Kit Protocol
Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix .
It is not necessary to remove mineral oil or kerosene .
If pH indicator I has beein added to Buffer PB , check that the color of the mixture is yellow .
If the color of the mixture is orange or violet , add 10 µl of 3 M sodium acetate , pH 5.0 , and mix .
The color of the mixture will turn to yellow .
Place a QIAquick spin column in a provided 2 ml collection tube .
To bind DNA , apply the sample to the QIAquick column and centrifuge for 30–60 s . Discard flow - through .
Place the QIAquick column back into the same tube .
To wash , add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s .
Discard flow - through and place the QIAquick column back in the same tube .
Centrifuge the column for an additional 1 min .
IMPORTANT : Residual ethanol from Buffer PE will not be completely removed unless the flow - through is discarded before this additional centrifugation .
Place QIAquick column in a clean 1.5 ml microcentrifuge tube .
To elute DNA , add 50 µl Buffer EB ( 10 mM Tris·Cl , pH 8.5 ) or water ( pH 7.0–8.5 ) to the center of the QIAquick membrane and centrifuge the column for 1 min .
Alternatively , for increased DNA concentration , add 30 µl elution buffer to the center of the QIAquick membrane , let the column stand for 1 min , and then centrifuge .
IMPORTANT : Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA .
The average eluate volume is 48 µl from 50 µl elution buffer volume , and 28 µl from 30 µl elution buffer .
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 2 ( page 15 ) to identify the dyes according to migration distance and agarose gel percentage and type .
