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7 v" _4 m' w0 H/ f* CBisulfite genomic sequencing - Protocol
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• 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.
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( { t% M: H; w6 Y5 g/ u• Phenol extract and ethanol precipitate the DNA and resuspend in either 10ul or 25ul volume.( i' w! o) k$ i& s6 J* T5 I8 {
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• 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)$ y$ O! O8 V! b" G0 N9 |
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• Add ca. 100 ng - 1 ug of digested DNA into the siliconised tube and make up to 25 ul with TE buffer.7 ?+ ~9 O2 p/ e! P! s
1 i+ k& F2 ?! _: q- f5 b. `0 i• 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
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6 f. M/ I5 u) Z, C5 |5 Q, G& ~• 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.+ T! C7 a$ A3 B! E3 K7 d
; k, x) _+ X" I, ]% e! l) r7 p: E7 A• add 270 uL of the freshly prepared sodium bisulfite/hydrochinone solution to the DNA.
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• Mix and overlay with 200 ul mineral oil. Incubate for 5 hours in a 55C heat block in the dark (cover with tin foil). , T/ ]9 P3 `; O! ]- \: I- D
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• 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.
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• Add 900 ul isopropanol to each, mix and centrifuge max speed for 20 mins at room temp.5 c# k1 O) j3 S/ ~" u
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• Discard supernatant, add 800 ul 70% EtOH, mix and spin for 15 min
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• Discard supernatant, remove last traces of EtOH with the pipette and air dry the pellet for 10 min.4 j4 h( a4 p& J+ {3 }3 v9 Z" u
. j0 }7 H" |6 u9 T( q$ P9 o• Resuspend in 25ul TE buffer, mix well." E9 Z) [8 X0 y6 o8 L3 \5 B4 |8 Y
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• Desulfonate the DNA by adding 2.5 ul 3M NaOH and incubating at 37C for 15 mins
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+ M4 v) s: {3 Q( x2 b& J• 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).
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1 s9 T0 T) V `% j. [• Wash pellet in 70% ethanol and then air dry (as described above) and resuspend in 25 ul TE buffer.
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9 q, k5 ~; t* _$ U* b: t, i• Use 2-5 ul in a PCR reaction.: `5 `- N0 [" q) U d+ F2 v b
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NOTES for PCR
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. Q0 L* x5 M; k& N$ j• try to amplify across a region no more than 300 bp." g2 h! ]% o f7 w" r% R( w
• Primers length about 20 bp
# q1 ^0 _, s0 \. c8 @& R1 X• When designing primers use WORD to convert all Cs to T except for those in CG dinucleotide. Then design primers .( i( H, } ~7 L" {) U
• Design primers WITHOUT CG
+ E/ W5 A5 J* E3 ?; s- m• Usually amplification best at 45C annealing- but not always
# U; z$ W4 c' @• May have to do nested PCR for best amplification/ h) z3 w* }$ R, [
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Special PCR buffer (10x):
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166 mM (NH4)2SO49 X! b+ p: J, i4 P
670 mM Tris pH 8.8
/ [( s) h$ ?5 q+ k9 \, G0 p$ ~67 mM MgCl2
/ M+ { e5 s, I, x8 d+ W S100 mM beta-mercaptoethanol( z+ g+ E+ V9 t9 \: I' U6 [
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