Qiagen Gel Extraction
Updated 7/19/2019 04:26 PM
Introduction
Materials
- Qiagen Gel Extraction Kit
- Buffer QG
- Sodium Acetate (maybe in step 4)
- Isopropanol
- Buffer PE
- Spin column and 2mL collecting tube
- Centrifuge
Procedure
- Excise the DNA fragment from the agarose gel with a scalpel.
- Weigh the gel slice in a colerless tube. add 3 volumes of Buffer QG to 1 volume gel ( 100mg gel ~ 100µL). The maximum amount of gel slice per spin column is 400mg. For >2% agarose gels,add 6 volumes Buffer QG.
- Incubate at 50 oC for 10 mins (or until the gel slice has completly dissolved). Vortex the tube every 2-3 mins during incubation to help dissolve the gel.
- After the gel slice has dissolved completely, check the color of the mixture is yellow (similar to Buffer QG withought dissolved agarose). If the color of the mixture is orange or violet, add 10µL 3M 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 by inverting.
- Place a *spin column in a 2mL collection tube, or into a vucuum manifold.
- Apply sample to the column and centrifuge for 1 min or apply vacuum until the sample has passed through the column. Discard flow-through and place the column back into the same collection tube.
- Add 500µL Buffer QG to the column and centrifuge for 1 min or apply vacuum. Discard the flow-through and place the column back into the same collection tube.
- Add 750µL Buffer PE to column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the column back into the same collection tube.
NOTE: if the DNA will be used for salt-sensitive applications, such as direct sequencing and blunt-ended ligation, let the column stand for 2-3 mins after addition of Buffer PE.
- Centrifuge the column in a 2mL collecction tube for 1 min. Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
- place each column innto a 1.5mL microcentrifuge tube. To elute DNA, add 10µL Buffer EB (
After running a gel, you still might have to use those bands!