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- Cut the gel to separate analytical and extraction gel; place analytical gel in UV illuminator.
- Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image and shut OFF the UV.
- Cut extraction gel under white light; avoid UV illuminating the extraction gel as this drastically decreases the DNA yield. If necessary, stain with Methyl Blue.
- Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 400mg per tube
- Add 3 volumes (6 volumes if you are afraid of getting a low yield) of Buffer QG to 1 volume of gel (100mg ~ 100ul)
- Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
- Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
- Add 1 gel volume of isopropanol
- Add max of 770ul to QIAquick column and centrifuge for 1 min (max speed, ~13,000rpm, RT)
- Run flow-through over column one more time.
- After the second time, discard flow-through and place column back in tube.
- If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
- Add 500uL of Buffer QG to column and centrifuge for 1 min (wash).
- Wash: add 0.75ml Buffer PE (make sure that the buffer has ethanol added to it) to column. Let stand for 2-5 minutes and then centrifuge for 1 min
- Discard flow-through & centrifuge for 1 min
- Place column into clean Eppendorf tube
- Add 50ul Buffer EB or water to center of membrane. Make sure to use warm EB (50C). (Use 30uL if worried about low concentration.)
- Let stand at RT for 4 min
- Centrifuge for 1 min
- Measure the concentration using the UV spectrophotometer.
Pro Tips
- You don't need 2 lanes if you aren't putting your gel under UV light (the blue light and SYBR safe is fine)
- You can up the IPA to 1/4 of the total volume
- Warm EB (50 mL conical filled w/ water, plop the tube inside, put it in the heat block)
- Don't let it stand at room temperature, you can do it at 5 degrees (heat block)
Gel Extraction Protocol using QIAgen MinElute Kit:
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