Methylated DNA immunoprecipitation protocol ; o z; Z3 ] L n
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15.3.1.1 Cell Collection and Lysis % `# w1 o- o- a: g H J( V& M1. 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.; P. k; w# B9 p+ j4 J
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. 5 H9 v5 v" C; u( L3. Meanwhile, place the GenDNA digestion buffer at room temperature (RT) and the GenDNA proteinase K on ice. $ v+ G( j0 q& _. G# j2 e2 t4. 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. # p. G$ X! M9 `: ~& f* B* V4 Y2 ~, V5. 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.8 T2 \' f- d$ H2 n0 X& z
6. 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. 1 E7 O8 M! w6 o" K6 i 7 t/ V d/ O1 _15.3.1.2 Extraction of Nucleic Acids and DNA Purification 2 y i$ N7 Z6 y7 R0 O" C, k( a1. 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.0 W! U# [" V% q, }' L0 e' q' O
2. Centrifuge at 1,700g for 10 min in a swinging bucket rotor. 4 |* i/ I ^* v9 v& r3. Transfer the aqueous (top) layer to a new tube. Increase volume if necessary (see above) and pipette slowly.+ G( U# a7 O; X, ?
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. ! X+ @/ M6 R4 _. d; J( J5. 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.# w9 p! x1 o* z) I
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., d7 C3 \1 ^1 u. @# k) q" P
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). 1 R" ]2 c0 |2 q7 N8. 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). - p8 C7 j/ o+ z7 o' r4 B% |9. For DNA analysis, run samples in a 1% agarose gel along with DNA size marker to visualize the DNA preparation efficiency.4 C" l* L$ A5 M( R- s3 {0 y, u: L
' I4 ?' z7 Q0 ?' R6 Q9 Z+ g0 ?15.3.1.3 DNA Shearing0 |7 F+ z- f2 f0 L. u1 E" E
1. In a 1.5 mL tube, dissolve the DNA sample in TE to reach 0.1 μg/μL. . X3 K# m: Y9 P- L( E2. Use a final volume of 300 μL of DNA sample in 1.5 mL tubes.4 i' Y$ o X1 ]9 h
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. 2 Q% m2 Q8 U. c: S7 ^9 H3 {! B4. Sheared DNA can be analyzed on agarose gel. 8 K) D W: w0 H. R4 n7 ` 6 P6 r7 j- d. F2 J作者: cronos0910 时间: 2011-12-27 00:26
8 T: m7 b. s8 b7 g• Digest ca. 1-2 ug genomic DNA with a restriction enzyme outside the region of interest. This will reduce viscosity of DNA and facilitate DNA degradation. 8 V9 y8 c( i3 K) w- K6 A5 R) u7 H0 o2 i) ~
• Phenol extract and ethanol precipitate the DNA and resuspend in either 10ul or 25ul volume. & }! r( O' c: u8 p 3 x5 t9 }7 ^+ l; O8 f6 R• Prepare siliconised screw-capped tubes (2mls): rinse tubes with dimethyldichlorosilane solution, let it dry for 10 min and rinse with distilled water (in the fume hood)4 O# L# Q) j2 z) K k
2 `$ X9 }6 K; h5 }# }• Add ca. 100 ng - 1 ug of digested DNA into the siliconised tube and make up to 25 ul with TE buffer. ) \$ O6 r. e" B/ t: R0 X% n2 w 0 e0 m8 T' W& N* J8 D8 d. s• Prepare bisulfite solutions: mix 3.8 g sodium bisulfite with 5 ml of water and 1.5 ml of 2 M NaOH and mix gently (don't use a magnetic stirrer, better a rotation platform!) in a dark tube. Dissolve 110 mg hydrochinone in 1 ml of water at 50C for 10 min. Add hydrochinone solution to the bisulfite solution and shake gently. Check that pH = 5.0$ Z7 c/ O P5 S5 M) c! k+ X
6 |& P* p, H. Z% j. e2 Z9 Y/ _• Boil DNA for 5 mins and then add 2.5uL of fresh 3M NaOH. Mix and incubate at 37C for 20 mins. This denatures the DNA. 2 E$ ?% h. |* p ' g& N& n4 d* i2 ^* M y. t• add 270 uL of the freshly prepared sodium bisulfite/hydrochinone solution to the DNA. Q$ q" y8 G/ R$ {( V
8 j+ k' R2 m! W$ W• Mix and overlay with 200 ul mineral oil. Incubate for 5 hours in a 55C heat block in the dark (cover with tin foil). 2 B) p8 k- e' A. @
4 D0 B& E- _! E k• Prepare 2 ml tubes each containing 600 ul water, 90 ul 3 M NaOAc and 2.5 ul glycogen. Transfer the DNA solution (possibly without mineral oil!) to the 2 ml tubes and mix. ( ^. Q; B$ h3 k5 q % P5 U8 y4 u; |& U: K• Add 900 ul isopropanol to each, mix and centrifuge max speed for 20 mins at room temp. * F, `$ \. c. E; W/ d k% T# _6 R. Z' F4 ~. }, s4 C5 z8 S+ ]8 n
• Discard supernatant, add 800 ul 70% EtOH, mix and spin for 15 min % X& w) H R+ G' m3 ^ / J p3 Q2 W, B7 A• Discard supernatant, remove last traces of EtOH with the pipette and air dry the pellet for 10 min.3 V; |/ Y2 Z5 g B' N( D
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• Resuspend in 25ul TE buffer, mix well. % ~/ a) i, h$ D \ 5 p8 e! q9 c. @• Desulfonate the DNA by adding 2.5 ul 3M NaOH and incubating at 37C for 15 mins 3 X# T; ^% O4 ^! ?4 y& N: ~& F
• Precipitate DNA by adding 32.5 ul 5 M ammonium acetate (pH 7.0) + 180ul ethanol. At this stage the sample can be kept in the fridge over night. Mix and centrifuge for 30 mins at 4C (cold room).' ?; z6 [1 ^# \/ H; I2 C# z* @$ D( o8 w
4 ^3 D- L6 y; |1 H$ U' V: O• Wash pellet in 70% ethanol and then air dry (as described above) and resuspend in 25 ul TE buffer.( m! c, ^( L! S! A" F0 F- o7 h
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• Use 2-5 ul in a PCR reaction.: c. j3 O1 A; M! X; K P: l
( b8 G0 \3 i7 B ( J9 Y7 y0 f1 D% SNOTES for PCR 6 q5 Z9 Y' Y5 |- m8 A6 C! }# u* M2 D; R$ {3 c4 j! x
• try to amplify across a region no more than 300 bp. 3 h" O6 j" s+ r9 m3 O7 q+ J/ \• Primers length about 20 bp 0 U. K$ B8 Z' J3 g4 _' d• When designing primers use WORD to convert all Cs to T except for those in CG dinucleotide. Then design primers .' F+ X- T6 Y# {$ j/ j! V8 z
• Design primers WITHOUT CG : F- k0 i0 `4 s6 ?. t* g• Usually amplification best at 45C annealing- but not always 9 i/ G& j* s5 \- `4 `1 u @7 [• May have to do nested PCR for best amplification ( R. c! x- j% L$ o2 E% q) f* e0 J* g" q9 t4 m8 Z# ^. e; n
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Special PCR buffer (10x): ! \; D0 b& p; v8 d+ e) N% _* A h8 A; Y* D$ T, B9 d! K! \. y
166 mM (NH4)2SO4 a8 D, x! a4 e4 ~
670 mM Tris pH 8.8 @6 P0 n& ]! z8 f/ a/ J; i! V67 mM MgCl23 }" ^8 {7 Q+ j+ l! V5 t
100 mM beta-mercaptoethanol @/ s5 a, o1 h9 s$ v, f 作者: cronos0910 时间: 2011-12-27 00:31
回复 cronos0910 的帖子 9 k! h, ]; y7 C 4 b7 G& p' D' g: p如果还有问题,欢迎提出,能帮得上忙的小弟一定帮,我叫威廉,在epigenetic这方面做过许多的研究作者: cronos0910 时间: 2011-12-27 00:37