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Jie:LabNotes/CpgSeq/2008-8-15
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==2rd Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit == {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|''' ''' | 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''' |- |1||BJ||178ng/ul x 2 tubes||11.2ul||8.8ul||130ul||43.3ng/ul x 20ul||2.18/2.97 |- |2||BJ_iPS_11||140ng/ul x 2 tubes||14.3ul||5.7ul||130ul||65.6ng/ul x 20ul||2.19/2.91 |- |6||IMB90||50ng/ul||20ul||0ul||130ul||22.7ng/ul x 10ul||2.05/2.78 |- |9||Hybrid2||200ng/ul||10ul||10ul||130ul||174.5ng/ul x 10ul||2.3/3.0 |- |12||Hues8||178.5ng/ul||13ul||7ul||130ul||8ng/ul x 10ul||1.37/1.01 |- |13||Hues42||20ng/ul x 2 tube||20ul||0ul||130ul||14ng/ul x 20ul|| |} *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 system== {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|''' ''' | 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''' |- |1||BJ||43.3ng/ul x 20ul||1ul||2+1+1+5ul||3+1.5+4.5||5+1.5+2.5||4+1.5+3.5 |- |2||BJ_iPS_11||65.6ng/ul x 20ul||1ul||2+1+1+5ul||3+1.5+4.5||5+1.5+2.5||4+1.5+3.5 |- |3||BJ-iPS_12||43.3ng/ul x 20ul||1ul||2+1+1+5ul||3+1.5+4.5||5+1.5+2.5||4+1.5+3.5 |- |6||IMB90||22.9ng/ul x 30ul||1ul||4+1+1+3ul|| || || |- |9||Hybrid_2||174.5ng/ul x 10ul + 65.6 x 10ul||1ul||2+1+1+5ul||3+1.5+4.5||5+1.5+2.5||4+1.5+3.5 |- | ||positive control||200ng/ul||1ul||1+1+1+6ul||1+1.5+6.5||2+1.5+5.5||1.5+1.5+6 |- | ||negative control||0||1ul||1+1+1+6ul||1+1.5+6.5||2+1.5+5.5||1.5+1.5+6 |} 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 0.2ul (10mM)0.02ul x300 174ul 30ul 30ul 60ul 6ul 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(08/16/2008)== x8 Template 10ul 2X iProof Mastermix 50ul 400ul AmpF6.2SoL (10uM) 4ul 32ul AmpR6.2SoL (10uM) 4ul 32ul 50X SYBG I 0.8ul 6.4ul H2O 31.2ul 249.6ul 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold. [[Image:20080817_PCR_No1_No2.jpg]]20080817_PCR_No1_No2 [[Image:20080817_PCR_No3_No9.jpg]]20080817_PCR_No3_No9 since the cpg30kV2.2 were not amplified, I redo the bisulfite conversion and capture of cpg30kV2.2. ==3rd Bisulfite conversion of DNA using Zymo EZ DNA Methylation-Gold kit(2008-08-29)== {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|''' ''' | 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''' |- |1||BJ||178ng/ul||11.2ul||8.8ul||130ul||48.1ng/ul x 10ul |- |2||BJ_iPS_11||140ng/ul||14.3ul||5.7ul||130ul||74.8ng/ul x 10ul |- |3||BJ_iPS_12||40ng/ul||20ul||0ul||130ul||41.4ng/ul x 10ul |- |9||Hybrid2||200ng/ul||10ul||10ul||130ul||122.1ng/ul x 10ul |- | ||Jurkat||154ng/ul x 2 tube||10ul||10ul||130ul||201.4ng/ul x 20ul |} *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 system== {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|''' ''' | 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+v2.2(21ng/ul) vol+H2O''' |- |1||BJ||48.1ng/ul x 10ul||1.5ul||10+1.5+2.5 |- |2||BJ_iPS_11||74.8ng/ul x 10ul||1.5ul||10+1.5+2.5 |- |3||BJ-iPS_12||41.4ng/ul x 10ul||1.5ul||10+1.5+2.5 |- |9||Hybrid_2||122.1ng/ul x 10ul||1.5ul||10+1.5+2.5 |- | ||positive control_Jurkat||201.4ng/ul||1.5ul||2+1.5+5.5 |- | ||negative control||0||1.5ul||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 0.2ul (10mM)0.02ul x300 174ul 30ul 30ul 60ul 6ul 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(09/01/2008)== x6 Template 10ul 2X iProof Mastermix 50ul 300ul AmpF6.2SoL (10uM) 4ul 24ul AmpR6.2SoL (10uM) 4ul 24ul 50X SYBG I 0.8ul 4.8ul H2O 31.2ul 187.2ul 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 10-> 72C 5 min -> 15C hold. [[Image:20080901_cpg30kV2.2_captured PCR_No_1_2_3_9_Jurkat.jpg]]20080902_cpg30kV2.2_captured PCR_No1_2_3_9_Jurkat [[Image:20080901_Quantification of No1_2.jpg]]20080901_quantification of No_1_2 [[Image:20080901_quantification of No_3_9.jpg]]20080901_quantificatio of No3_9 yield: No_1: cpg30k + suppressor: 6ng/ul x 15ul V2.1: 6ng/ul x 15ul V2.2: 4ng/ul x 15ul V2.3: 6ng/ul x 15ul V2.2_09/01/08: 3ng/ul x 15ul mix 5ul cpg30k + suppressor + 5ul V2.1 + 10ul V2.2_09/01/08 + 5ul V2.3 = 4.8ng/ul x 25ul No_2: cpg30k + suppressor: 6ng/ul x 15ul V2.1: 6ng/ul x 15ul V2.2: 3ng/ul x 15ul V2.3: 5ng/ul x 15ul V2.2_09/01/08: 4ng/ul x 15ul mix 5ul cpg30k + suppressor + 5ul V2.1 + 7.5ul V2.2_09/01/08 + 6ul V2.3 = 5ng/ul x 23.5ul No_3: cpg30k + suppressor: 4.5ng/ul x 15ul V2.1: 4.5ng/ul x 15ul V2.2: 2.5ng/ul x 15ul V2.3: 2.5ng/ul x 15ul V2.2_09/01/08: 2ng/ul x 15ul mix 5ul cpg30k + suppressor + 5ul V2.1 + 4ul V2.2 + 9ulV2.3 + 5ul V2.2_09/01/08 = 3.2ng/ul x 28ul No_9: cpg30k + suppressor: 5ng/ul x 15ul V2.1: 3ng/ul x 15ul V2.2: 1.5ng/ul x 15ul V2.3: 2ng/ul x 15ul V2.2_09/01/08: 3ng/ul x 15ul mix 4ul cpg30k + suppressor + 7ul V2.1 + 10ul V2.3 + 7ul V2.2_09/01/08 = 2.8ng/ul x 28ul ==sequencing library construction== ==2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 == reaction system x4 H2O 42.6ul 170.4ul 2x Master mix 50ul 200ul dUTP(1mM) 2ul 8ul AmpF6.3(10uM) 2ul 8ul AmpR6.3(10uM) 2ul 8ul 50x SYBG I 0.4ul 1.6ul template(4ng/ul) 0.5ul 2ul Total 100ul 400ul 94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Bioneer column purification. Yield: No_1: 33.2ng/ul x 30ul No_2: 39.4ng/ul x 30ul No_3: 56.7ng/ul x 30ul No_9: 49.4ng/ul x 30ul ==Digestion with MmeI== No_1_2_3_9 x4 Total 30ul dUTP_PCR 20ul 10X NEBuffer 4 4ul 16 1mM SAM(fresh) 4ul 16 2U/ul Mme I 8ul 32 ddH2O 4ul 16 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. MinEulte column purification. Elute in 12ul EB. [[Image:20080901_dUTP_PCR and MmeI.jpg]]20080901_dUTP_PCR and MmeI ==step3. USER digestion== DNA 10ul USER 3ul total 13ul 37C 12h. ==step4. S1 nuclease digestion== No1_2_3_9 x4 10 x S1 nuclease buffer: 2ul 8ul DNA after USER digestion: 13ul 13ul S1 nuclease (10U/ul): 1ul 4ul ddH2O 4ul 16ul 37C 10mins. Minelute cloumn purify. Elute in 16ul H2O. ==step5. end repair== No1_2_3_9 positive control x5 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 12.5 dATP 2.5ul 2.5ul 12.5 10xendrepair buffer 2.5ul 2.5ul 12.5 enzyme 0.5ul 0.5ul 2.5 extra ATP(10mM) 2.5ul 2.5ul 12.5 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O. ==step6. adapter ligation== No1_2_3_9 positive control negative control x6 DNA 13ul 13ul ddH2O 13ul 48 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 3 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul 3 2xQuickLiage buffer 15ul 15ul 15ul 90 QuickLigase enzyme(NEB) 1ul 1ul 1ul 6 extra ATP(10mM) 2.5ul 2.5ul 2.5ul 15 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O. TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O. [[Image:20080902_gul size selectin after adaptor ligation.jpg]]20080902_gel size selection after adaptor ligation [[Image:20080902_positive and negative control of adaptor ligation.jpg]]20080902_gel of control and genative control ==step7. Nick-translation== set up the ligation system: x6 Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 12ul 10mM dNTP 0.4ul 2.4ul 1mg/ml BSA 2ul 12ul Bst polymerase(8U/ul) 1ul 6ul 65C for 25 minutes -> keep on ice. ==step8. PCR of sequencig library== x6 Nick-translated DNA 20ul Solexa_PCR_up(10uM) 2ul 12ul Solexa_PCR_lo(10uM) 2ul 12ul 2xiProof master mix 50ul 300ul 50x SYBG 0.8ul 4.8ul ddH2O 25.2ul 151.2ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. [[Image:20080902_sequencing library of No1_2_3_9.jpg]]20080902_sequencing library of No1_2_3_9 Qiaquick column purification. Yield: No_1: BJ, 17.4ng/ul (117nM) x 30ul No_2: BJ_iPS_11, 18.6ng/ul (125nM) x 30ul No_3: BJ_iPS_12, 32.7ng/ul (220nM) x 30ul No_9: Hybrid_2, 28.3ng/ul (190nM) x 30ul Sent 15ul of each sequencing library to Yuan Gao on 08-09-02. ==Blunt clone and transformation of captured PCR products and random sequencing library of No1_BJ (2008-09-04)== refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2008-9-4]
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