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Gel Extraction (QIAgen kit) 1. Excise gel band with as little excess agarose as possible. 2. Place gel slice in a 1.5mL tube and find the slice’s mass in milligrams. 3. Add 3 volumes of Buffer QG to the tube. (Assume mass of the slice in mg to be equal to volume in ul) 4. Incubate at 55 degrees, 10’. 5. Vortex and add a volume isopropanol equal to gel slice volume. 6. Vortex again and apply up to 800ul of the mixture to a QIAgen column/collection tube. Spin at 14,000rpm for 1’. Discard flow through. a. When applying the mixture, make sure to include any precipitates that form upon addition of isopropanol. b. If volume exceeds 800ul, repeat this step with the remainder of liquid before proceeding. 7. Add 500ul Buffer QG to the column and spin again at 14,000rpm for 1’. Discard flow through. 8. Add 750ul Buffer PE and let sit on bench top for 5’. Spin at 14,000rpm and discard flow through. 9. Add 350ul Buffer PE and let sit on bench top for 2’. Spin at 14,000rpm and discard flow through. 10. Spin the empty column/collection tube one more time at 14,000rpm to remove all traces of Buffer PE. 11. Take two 1.5mL microcentrifuge tubes for each sample, and cut the top off of one of them. 12. Remove QIAgen column from collection tube and place it in a topless 1.5mL tube. Discard collection tube. 13. Apply 30ul Buffer EB directly to the center of the column’s membrane. Let sit on bench top for 3’. Spin at 14,000rpm. (Topless tubes are used in this step because the tops tend to break off and shatter, and the pieces fly into other wells of the centrifuge.) 14. Transfer the eluate from the topless tube to the tube with a top and label the top. 15. Run 2ul of eluate on an agarose gel to quantify sample.
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