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Jie:LabNotes/CpgSeq/2009-1-22
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==6th Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit == {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|'''No''' | align="center" style="background:#f0f0f0;"|'''sample ''' |align="center" style="background:#f0f0f0;"|'''sample concentration''' | align="center" style="background:#f0f0f0;"|'''sample volumn''' | align="center" style="background:#f0f0f0;"|'''ddH2O ''' | align="center" style="background:#f0f0f0;"|'''conversion reagents''' | align="center" style="background:#f0f0f0;"|'''conversed DNA concentration and volumn''' | align="center" style="background:#f0f0f0;"|'''260:280/260:230''' |- |21||PGP1_iPS1||50ng/ul x 3 tubes||20ul||0ul||130ul||179.1ng/ul x 10ul||1.97/1.72 |- |22||PGP1_L||293ng/ul x 3 tubes||6.8ul||13.2ul||130ul||368.7ng/ul x 20ul||2.13/2.04 |- |23||PGP3_L||242ng/ul x 3 tubes||6.8ul||13.2ul||130ul||238.6ng/ul x 20ul||2.07/1.89 |- |24||PGP9_L||249ng/ul x 3 tubes||6.8ul||13.2ul||130ul||223.7ng/ul x 20ul||2.05/1.89 |- |25||PGP1F.2||25ng/ul x 4 tubes||20ul||0ul||130ul||84ng/ul x 10ul||1.67/1.2 |- |26||PGP9F.2||14ng/ul x 4 tube||20ul||0ul||130ul||35.1ng/ul x 10ul||1.2/0.66 |- |} *Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent. *Add 130ul of CT conversion reagent to samples. *98C 10min -> 64C 150min -> 4c overnight. *Add 600ul M-Binding buffer to spin columns. *Add the converted samples to the columns, close the gap and mix by inverting several times. *Spin at 15,000rpm for 30sec. *Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through. *Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec. *Add 200ul M-Wash Buffer, spin for 30 sec. *Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec. *Measure the DNA with Nanodrop: RNA ==set up the capture reaction== {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|'''No ''' | align="center" style="background:#f0f0f0;"|'''sample ''' |align="center" style="background:#f0f0f0;"|'''sample concentration''' | align="center" style="background:#f0f0f0;"|'''10xLigase buffer''' | align="center" style="background:#f0f0f0;"|'''template+cpg30k(50.7ng/ul)+suppressor(8nM each)vol+H2O''' | align="center" style="background:#f0f0f0;"|'''template+v2.1(21ng/ul) vol+H2O''' | align="center" style="background:#f0f0f0;"|'''template+v2.2(21ng/ul) vol+H2O''' | align="center" style="background:#f0f0f0;"|'''template+v2.3(20ng/ul) vol+H2O''' |- |21||PGP1_iPS1||179.1ng/ul x 10ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5 |- |22||PGP1_L||368.7ng/ul x 10ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5 |- |23||PGP3_L||238.6ng/ul x 10ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5 |- |24||PGP9_L||223.7ng/ul x 10ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5 |- |25||PGP1F.2||84ng/ul x 10ul||1ul||7+1+1+0ul|||||| |- |26||PGP9F.2||35.1ng/ul x 10ul||1ul||7+1+1+0ul|||||| |- | ||positive control||200ng/ul||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5 |- | ||negative control||0||1ul||1+1+1+6ul||1+1.5+6.5||3+1.5+4.5||2+1.5+5.5 |} dNTP concentration 10mM(NEB) ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP: 0.58ul 0.1ul 0.1ul 0.2 (10mM)0.02ul x100 58ul 10ul 10ul 20ul 2ul 95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h -> <BR>9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min -> <BR>add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold. ==PCR(01/26/2009)== x12 Template 10ul 2X iProof Mastermix 50ul 600ul AmpF6.2SoL (10uM) 4ul 48ul AmpR6.2SoL (10uM) 4ul 48ul 50X SYBG I 0.8ul 9.6ul H2O 31.2ul 374.4ul 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold. [[Image:20090126_No_21_22.jpg]]20090126_No_21_22 [[Image:20090126_No_23_24_25_26.jpg]]20090126_No_23_24_25_26 ==quantification of gel purified amplicon== no_21 PGP1_iPS1: cpg30k 1ng/ul x 10ul; V2.1 1ng/ul x 10ul; No_22 PGP1_L: cpg30k 3ng/ul x 10ul; No_23 PGP3_L: cpg30k 3ng/ul x 10ul; V2.1 3 ng/ul x 10ul; V2.2 1ng/ul x 10ul; V2.3 3ng/ul x 10ul; No_24 PGP9_L: cpg30k 3ng/ul x 10ul; V2.1 3 ng/ul x 10ul; V2.3 3ng/ul x 10ul; No_25 PGP1F.2: cpg30k 2ng/ul; No_26 PGP9F.2: cpg30k 2ng/ul; no_21 PGP1_iPS1: cpg30k 1ng/ul 2ul + V2.1 1ng/ul x 2ul; No_22 PGP1_L: cpg30k 3ng/ul x 2ul; No_23 PGP3_L: cpg30k 3ng/ul 1ul + V2.1 3ng/ul x 1ul + V2.2 1ng/ulx 2ul + V2.3 3ng/ul x 1ul; No_24 PGP9_L: cpg30k 3ng/ul 1ul + V2.1 3ng/ul x 1ul + V2.3 3ng/ul x 1ul; No_25 PGP1F.2: cpg30k 2ng/ul x 2ul; No_26 PGP9F.2: cpg30k 2ng/ul x 2ul; ==sequencing library construction== ===2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 === reaction system x14 x2 H2O 42.6ul 596.4ul 2x Master mix 50ul 700ul dUTP(1mM) 2ul 28ul AmpF6.3(10uM) 2ul 28ul AmpR6.3(10uM) 2ul 28ul 50x SYBG I 0.4ul 5.6ul template(4ng/ul) 0.5ul 2 uleach Total 100ul 1400ul 94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Bioneer column purification. Yield: No_21:40.5ng/ul; No_22:39.1ng/ul; No_23:35.6ng/ul; No_24:32.4ng/ul; No_25:47ng/ul; No_26:35.2ng/ul; No_21_2: 28.9ng/ul; ===Digestion with MmeI=== No_21~26 x6 Total 40ul dUTP_PCR 20ul 10X NEBuffer 4 4ul 24 1mM SAM(fresh) 8ul 48 2U/ul Mme I 8ul 48 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. MinEulte column purification. Elute in 12ul EB. ===step3. USER digestion=== DNA 10ul USER 3ul total 13ul 37C 2h. ===step4. S1 nuclease digestion=== No21~26 x7 10 x S1 nuclease buffer: 2ul 14ul DNA after USER digestion: 13ul 13ul S1 nuclease (10U/ul): 1ul 7ul ddH2O 4ul 28ul 37C 10mins. Minelute cloumn purify. Elute in 16ul H2O. ===step5. end repair=== No21~26 positive control x7 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 17.5 dATP 2.5ul 2.5ul 17.5 10xendrepair buffer 2.5ul 2.5ul 17.5 enzyme 0.5ul 0.5ul 3.5 extra ATP(10mM) 2.5ul 2.5ul 17.5 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O. ===step6. adapter ligation=== No21~26 positive control negative control x8 DNA 13ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 4 100uM Solexa_2_PE adaptor 0.5ul 0.5ul 0.5ul 4 2xQuickLiage buffer 15ul 15ul 15ul 120 QuickLigase enzyme(NEB) 1ul 1ul 1ul 8 extra ATP(10mM) 2.5ul 2.5ul 2.5ul 20 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB. TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O. ===step7. Nick-translation=== set up the ligation system: x8 Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 16ul 10mM dNTP 0.4ul 3.2ul 1mg/ml BSA 2ul 16ul Bst polymerase(8U/ul) 1ul 8ul 65C for 25 minutes -> keep on ice. ===step8. PCR of sequencig library=== x8 Nick-translated DNA 20ul Solexa_PCR_up(10uM) 2ul 16ul Solexa_PCR_PE_loH(10uM) 2ul 16ul 2xiProof master mix 50ul 400ul 50x SYBG 0.8ul 6.4ul ddH2O 25.2ul 201.6ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. [[Image:20090130_parallel Solexa Sequencing library No21~26.jpg]]20090130_parallel Solexa Sequencing library No21~26 Qiaquick column purification and quantification: No_21 PGP1_iPS1: 16.4 ng/ul(90.3nM) x 30ul; No_22 PGP1_L: 14.1 ng/ul(77.6nM) x 30ul; No_23 PGP3_L: 14.7 ng/ul(80.9nM) x 30ul; No_24 PGP9_L: 17.6 ng/ul(96.9nM) x 30ul; No_25 PGP1_F.2: 18.5 ng/ul(93.4nM) x 30ul; No_26 PGP9_F.2: 19.3 ng/ul(97.4nM) x 30ul;
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