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Purification of DNA from agarose gels
Lenzmeier Research Laboratory
Purification of DNA fragments from agarose gels using the Qiaquick Gel Extraction Kit Qiagen catalog # 28704 (50 reactions) or #28706 (250 reactions) Note: This DNA binding matrix effectively binds 70 bp to 10,000 bp DNA fragments 1. Excise the DNA fragment from the gel using a clean sharp scalpel. 2. Weigh the gel slice on a clean weigh boat. Cut up the gel slice into fragments that weigh no more than 300 mg (0.3 g) and transfer each gel slice into a new 1.5 ml microcentrifuge tube. 3. Add 3 volumes of QG buffer (yellow in color) and vortex briefly. For example: if your gel slice weighed 100 mg (0.1 g), add 300 ul; if your gel slice weighed 200 mg (0.2 g), add 600 ul. 4. Incubate tubes 10 minutes at 55oC to melt the agarose and dissolve the gel. To facilitate this process, vortex your sample every 2-3 minutes to ensure maximal resuspension. 5. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to QC without dissolved agarose). If it is yellow proceed. If it is very orange or even purple, add 10 ul of 3 M sodium acetate and vortex. If still orange or purple, add sodium acetate incrementally until the solution turns yellow. 6. Add 1 volume of 100% isopropanol (aka 2-propanol) and then vortex briefly to mix. For example: if your gel slice weighed 100 mg (0.1 g), add 100 ul; if your gel slice weighed 200 mg (0.2 g), add 200 ul. 7. Place the Qiaquick spin column into the provided 2 ml tube. Note: If you have more than 700 ul of dissolved agarose/QG/isopropanol, you will do steps 8-9 twice to run the entire solution through the tube 8. Add up to 700 ul of dissolved agarose/QG/isopropanol to the Qiaquick spin column and centrifuge 30-60 seconds at 13,000 rpm. 9. Remove the column, discard the liquid flow-through from the 2 ml tube down the drain and place the column back into the 2 ml tube. Note: Either return to step and run the remaining liquid through the column by repeating steps 8 and 9 or proceed to step 10 if all your sample has already been run through the column. 10. Add 700 ul of PE buffer to the column and centrifuge 30-60 seconds at 13,000 rpm. 11. Remove the column, discard the liquid flow-through from the 2 ml tube, and place the column back into the 2 ml tube. Page 1 of 2
Purification of DNA from agarose gels
Lenzmeier Research Laboratory
12. Centrifuge 60 seconds at 13,000 rpm to remove any residual PE buffer (Don’t add any liquid before this step). 13. Place the Qiaquick column into a new 1.5 ml microcentrifuge tube. Label tube so you know it is yours and what is inside the tube. 14. To elute (remove) DNA from the column, add 50 ul of EB buffer (10 mM Tris) to the center of the column. Let stand one minute on the bench top. 15. Centrifuge 60 seconds at 13,000 rpm to elute DNA from the column into the new microcentrifuge tube. 16. Remove column and cap your purified product. Make sure your tube is labeled well. You should have about 45 ul of sample. Note: You should seriously consider double-checking that your purification worked by analyzing your product on an agarose gel.