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Methylated DNA immunoprecipitation protocol
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! B: ^+ q: h; [" A! o15.3.1.1 Cell Collection and Lysis
L7 T; }! ]8 y1. Pellet suspension culture out of its serum-containing medium. Trypsinize adherent cells and collect cells from the flask. Centrifuge at 300g for 5 min at 4°C.( m- N" q" C3 X2 S; R
2. Discard the supernatant. Resuspend cells in 5–10 mL ice-cold PBS. Centrifuge at 500g for 5 min. Discard the supernatant. Repeat this resuspension and centrifugation step once more. This step is to wash the cells.
2 m: @9 A5 a% h4 z3 V3 u3. Meanwhile, place the GenDNA digestion buffer at room temperature (RT) and the GenDNA proteinase K on ice.: K9 f, W4 G* S+ a
4. Add GenDNA proteinase K to the GenDNA digestion buffer before use. The stock of provided proteinase K is 200X; e.g., add 5 μL per 1 mL of digestion buffer, i.e., the freshly prepared complete digestion buffer to be used directly." V5 W/ B" E- ^7 ?' ]
5. Resuspend cells in complete digestion buffer (1 volume). For 3 million cells, use 300 μL complete digestion buffer. For 10 million cells, use 500 μL complete digestion buffer. It might be necessary to use more buffer to avoid problems when performing the extractions below. If necessary, for 3 million cells, use up to 600 μL of buffer. For 10 million cells, use up to 1,000 μL of buffer.
J; n4 V0 s6 g3 x( ]+ ~% `0 I6. Cell lysis: Incubate the samples with shaking at 50°C for 12–18 h in tightly capped tubes. That is the cell lysis step. At this stage, samples are viscous. After 12 h incubation the tissue should be almost indiscernible, a sludge should be apparent from the organ samples, and tissue culture cells should be relatively clear.# j' y5 u2 a) f6 L7 m9 H" C
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15.3.1.2 Extraction of Nucleic Acids and DNA Purification
8 ~6 j5 P4 e5 ^. F1. Thoroughly extract the samples with an equal volume of phenol:chloroform:isoamyl alcohol. Add 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1). One volume is about 500 μL. It is possible to incubate the samples at RT for 10 min on a rotating wheel before centrifugation. Use gentle rotation and do not vortex. Work under a fume hood.) \- L! o' w" ?7 ]5 w3 f! q
2. Centrifuge at 1,700g for 10 min in a swinging bucket rotor.
! a0 N( B8 K+ `! n! n3. Transfer the aqueous (top) layer to a new tube. Increase volume if necessary (see above) and pipette slowly./ ~5 n8 A7 a: @( j; y
4. Add 1/2 volume of GenDNA precipitant and 2 volumes of 100% ethanol (see Note 2). That is to purify the DNA. One volume is about 500 μL and corresponds to the original amount of top layer. Add therefore 250 μL of precipitant and 1,000 μL of 100% ethanol. The DNA should immediately form a stringy precipitate.$ }- u8 b8 k' S& j
5. Recover DNA by centrifugation at 1,700g for 2 min. This brief precipitation in the presence of an optimized high salt precipitant (GenDNA precipitant) reduces the amount of RNA in the DNA sample. For long-term storage, it is convenient to leave the DNA in the presence of ethanol.: G* `, l: o7 b( Z5 b
6. Rinse the pellet with 70% ethanol. Decant ethanol and air-dry the pellet. It is important to rinse extensively to remove any residual of salt and phenol.3 _& [. W0 V/ `( D& S0 M
7. Resuspend the pellet of DNA at ～1 mg/mL in GenDNA TE until dissolved. Shake gently at room temperature or at 65°C for several hours to facilitate solubilization. Store at 4°C. From 3 million cells, ～20–30 μg of DNA can be expected in a volume of 20–30 μL. From 10 million cells, ～50–100 μg of DNA can be expected (in a volume of 200–300 μL). If possible, it is recommended to get at least 30 μg of DNA (when enough material is available) to be able to work with 30 μg of DNA (see Section 3.1.3).- d8 {" \8 I: v& s( ~) }! s' l0 d
8. If necessary, residual RNA can be removed at this step by adding 2 μL of GenDNA RNase (DNase-free) per milliliter of DNA sample and incubating 1 h at 37°C, followed by phenol:chloroform extraction and ethanol precipitation (similar to above).6 K) ?+ X" ~- u( z ?; K* \ Q7 _
9. For DNA analysis, run samples in a 1% agarose gel along with DNA size marker to visualize the DNA preparation efficiency.
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1 }3 w+ S, P, z/ i15.3.1.3 DNA Shearing
; K. X+ [+ b1 W$ g$ G( d t; c1. In a 1.5 mL tube, dissolve the DNA sample in TE to reach 0.1 μg/μL.* p3 }( i5 [; A1 r. h
2. Use a final volume of 300 μL of DNA sample in 1.5 mL tubes.
* K1 e& @4 b8 {: g# G" y6 @, M6 @( @3. Shear the DNA using the Bioruptor: at “LOW” power using the following cycles: (15 s “ON” and 15 s “OFF”) for a total time of 10 min.
. M6 o6 e% Y0 u( {" k" d% U2 d% K4. Sheared DNA can be analyzed on agarose gel.
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发表于 2011-12-23 08:42
