Extraction of genomic DNA
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[MATERIALS] - [INDEX]
Procedure
- Grow two 3 L flasks of Dicty cells.
- Harvest cells by centrifugation at 3K for 10 min.
- Wash cells with Buffer 3, pellet as in step #2.
- Repeat wash 3 times
- Following the last spin wipe all buffer from the tubes and resuspend the cells in 100 ml of Buffer 2 (Triton Buffer).
Add an additional 700 µl of Triton when cells have lysed.
- Incubate solution on ice for 10 minutes with occasional swirling.
- Pellet the nuclei at 5K for 5 minutes. The pellet should appear as a white solid with a dark center.
- Resuspend the pellet in 50 ml of Buffer 1 (NO TRITON), being careful not to bring along the darker material.
- Pellet at 5K for 5 minutes.
- Repeat wash if alot of dark stuff is present.
- Resuspend the pellet in 6.0 ml water then add 8.0 ml 0.25 M EDTA (the total
volume should be about 16 ml).
- Gently mix the solution and add 4.0 ml 20% Sarcosyl.
It is very important to handle this solution carefully. Do not shake vigorously or the DNA will become
sheared.
- Adjust the total volume of the solution to 20 ml, add it to a 250 ml flak and incubate it
at 65°C for 5-10 minutes.
Swirl the solution, it should become viscous.
- Add 20g CsCl add mix gently. It may take a while to get this in solution but be patient.
- Once the CsCl has dissolved, add 2.0 ml of Ethidium Bromide (10 mg/ml) and load into
two ultracentrifuge quick seal tubes.
The solution should be viscous, so be careful not to glob it all over.
- Centrifuge for 24 hr at 50K.
- Recover the pink band, extract the EthBr with salt saturated isopropanol, and dialyze the
DNA against TE.
- To concentrate, ethanol precipitate.
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Materials
- Buffer 1 (Extraction Buffer)
- 50 mM Tris; pH 8.0
- 10 mM NaCl
- 3 mM MgCl2
- 3 mM CaCl2
- 0.5 M Sorbitol
- Buffer 2 (Triton Extraction Buffer)
- Buffer 1 containing 0.6% Triton
- Buffer 3 (Dicty Wash Buffer)
- 0.2% NaCl
or use DB, or PDF
- 20% Sarcosyl
- 0.25 M EDTA
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