Gel Extraction
From 2006.igem.org
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[[McGill_University_2006|Home]] <br> | [[McGill_University_2006|Home]] <br> | ||
[[Protocols|Protocols]] | [[Protocols|Protocols]] | ||
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| + | 1. Cut DNA band from agarose gel with scalpel blade, removing as much agarose as possible. Place gel piece in a tared 1.5 mL Epi tube. <br> | ||
| + | 2. Weigh tube with gel piece in it and calculate weight of agarose gel. If weight is greater than 250 mg, cut the gel into pieces of less than 250 mg each, and place in separate 1.5 mL Epi tubes. <br> | ||
| + | 3. If DNA fragment is between 100 bp and 4 kb, add 3 volumes of Buffer QX1 to each tube (eg. if tube contains 250 mg agarose, add 750 uL of Buffer QX1). <br> | ||
| + | If DNA fragment is less than 100 bp, add 6 volumes of Buffer QX1 to each tube.* <br> | ||
| + | If DNA fragment is greater than 4 kb, add 3 volumes of Buffer QX1 and 2 volumes of H2O to each tube.* <br> | ||
| + | If agarose gel is greater than 2%, add 6 volumes of Buffer QX1.* <br> | ||
| + | NB. for cases marked with a *, 250 mg may be too large a piece to process in a single tube, and should be split up into smaller portions. <br> | ||
| + | 4. Resuspend QIAEX II beads by vortexing for 30 sec. <br> | ||
| + | 5. If gel portion contains less than 2 ug of DNA, add 10 uL of QIAEX II. <br> | ||
| + | If gel portion contains between 2 and 10 ug of DNA, add 30 uL of QIAEX II. <br> | ||
| + | If gel portion contains more than 10 ug of DNA, add 30 uL of QIAEX II for each 10 ug of DNA (eg. if 20 ug of DNA, use 60 uL of QIAEX II). <br> | ||
| + | 6. Mix by vortexing. <br> | ||
| + | 7. Incubate at 50 C for 10 min, vortexing every 2 min to keep beads suspended. <br> | ||
| + | NB. solution colour must remain yellow during this stage. If it is orange or purple, add 10 uL of 3M sodium acetate and mix to return colour to yellow. If sodium acetate is added, incubation must continue for at least an additional 5 min. <br> | ||
| + | 8. Centrifuge tubes for 30 sec, remove supernatant with pipet. <br> | ||
| + | 9. Add 500 uL of Buffer QX1 to each tube, resuspend pellet by vortexing, then centrifuge for 30 sec and remove supernatant with pipet. <br> | ||
| + | 10. Add 500 uL of Buffer PE to each tube, resuspend pellet by vortexing, then centrifuge for 30 sec and remove supernatant with pipet. <br> | ||
| + | 11. Repeat step 10. <br> | ||
| + | 12. Air-dry pellet for 10 to 15 min until it becomes white. <br> | ||
| + | 13. Add 20 uL of H2O, resuspend pellet by vortexing. <br> | ||
| + | 14. If DNA fragment is less than 4 kb, incubate tubes at room temperature for 5 min. <br> | ||
| + | If DNA fragment is between 4 and 10 kb, incubate tubes at 50 C for 5 min. <br> | ||
| + | If DNA fragment is greater than 10 kb, incubate tubes at 50 C for 10 min. <br> | ||
| + | 15. Centrifuge tubes for 30 sec. Pipet supernatant into clean tube, and preserve supernatant which contains purified DNA. | ||
Latest revision as of 18:28, 13 July 2006
1. Cut DNA band from agarose gel with scalpel blade, removing as much agarose as possible. Place gel piece in a tared 1.5 mL Epi tube.
2. Weigh tube with gel piece in it and calculate weight of agarose gel. If weight is greater than 250 mg, cut the gel into pieces of less than 250 mg each, and place in separate 1.5 mL Epi tubes.
3. If DNA fragment is between 100 bp and 4 kb, add 3 volumes of Buffer QX1 to each tube (eg. if tube contains 250 mg agarose, add 750 uL of Buffer QX1).
If DNA fragment is less than 100 bp, add 6 volumes of Buffer QX1 to each tube.*
If DNA fragment is greater than 4 kb, add 3 volumes of Buffer QX1 and 2 volumes of H2O to each tube.*
If agarose gel is greater than 2%, add 6 volumes of Buffer QX1.*
NB. for cases marked with a *, 250 mg may be too large a piece to process in a single tube, and should be split up into smaller portions.
4. Resuspend QIAEX II beads by vortexing for 30 sec.
5. If gel portion contains less than 2 ug of DNA, add 10 uL of QIAEX II.
If gel portion contains between 2 and 10 ug of DNA, add 30 uL of QIAEX II.
If gel portion contains more than 10 ug of DNA, add 30 uL of QIAEX II for each 10 ug of DNA (eg. if 20 ug of DNA, use 60 uL of QIAEX II).
6. Mix by vortexing.
7. Incubate at 50 C for 10 min, vortexing every 2 min to keep beads suspended.
NB. solution colour must remain yellow during this stage. If it is orange or purple, add 10 uL of 3M sodium acetate and mix to return colour to yellow. If sodium acetate is added, incubation must continue for at least an additional 5 min.
8. Centrifuge tubes for 30 sec, remove supernatant with pipet.
9. Add 500 uL of Buffer QX1 to each tube, resuspend pellet by vortexing, then centrifuge for 30 sec and remove supernatant with pipet.
10. Add 500 uL of Buffer PE to each tube, resuspend pellet by vortexing, then centrifuge for 30 sec and remove supernatant with pipet.
11. Repeat step 10.
12. Air-dry pellet for 10 to 15 min until it becomes white.
13. Add 20 uL of H2O, resuspend pellet by vortexing.
14. If DNA fragment is less than 4 kb, incubate tubes at room temperature for 5 min.
If DNA fragment is between 4 and 10 kb, incubate tubes at 50 C for 5 min.
If DNA fragment is greater than 10 kb, incubate tubes at 50 C for 10 min.
15. Centrifuge tubes for 30 sec. Pipet supernatant into clean tube, and preserve supernatant which contains purified DNA.
