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Run Gel/ Gel extraction
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(New page: Gel Extraction Protocol using QIAquick Gel Extraction Kit: All centrifugation steps are done at speeds > 13000 rpm 1. Look at the gel under low wavelength UV (high wavelengths will denat...) |
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All centrifugation steps are done at speeds > 13000 rpm | All centrifugation steps are done at speeds > 13000 rpm | ||
Revision as of 17:20, 28 October 2008
Gel Extraction Protocol using QIAquick Gel Extraction Kit:
All centrifugation steps are done at speeds > 13000 rpm
1. Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image, cut out relevant bands and shut OFF the UV.
2. Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 400mg per tube
3. Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100ul)
4. Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
5. Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
6. Add 1 gel volume of isopropanol (not necessary for DNA fragments between 500-4000bp)
7. Add max of 770ul to QIAquick column and centrifuge for 1 min (max speed, ~13,000rpm, RT)
8. Discard flow-through and place column back in tube.
9. If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
10. Wash: add 0.75ml Buffer PE(make sure that the buffer has ethanol added to it) to column and centrifuge for 1 min
11. Discard flow-through & centrifuge for 1 min
12. Place column into clean Eppendorf tube
13. Add 50ul Buffer EB or water to center of membrane
14. Let stand at RT for 3 min
15. Centrifuge for 5 min
16. Measure the concentration using the UV spectrophotometer.
