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==parkinson's patient DNA sample capture by cpg97k== ===Bisulfite conversion of Parkinson's patient DNA === {| 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''' |- |Par_1||5028||320ng/ul x 1 tube||7ul||13ul||130ul||42.8ng/ul x 10ul |- |Par_2||4735||260ng/ulx 1 tubes||8ul||12ul||130ul||46.9ng/ul x 10ul |- |Par_3||4789||280ng/ulx 1 tubes||7ul||13ul||130ul||31.2ng/ul x 10ul |- |Par_4||5171||420ng/ulx 1 tubes||5ul||15ul||130ul||41.8ng/ul x 10ul |- |Par_5||1818||480ng/ulx 1 tubes||4.5ul||15.5ul||130ul||21.1ng/ul x 10ul |- |Par_6||1947||360ng/ul x 1 tubes||6ul||14ul||130ul||55ng/ul x 10ul |- |Par_7||1741||560ng/ul x 1 tube||4ul||16ul||130ul||27.3ng/ul x 10ul |- |Par_8||4977||260ng/ulx 1 tube||8ul||12ul||130ul||38.4ng/ul x 10ul |- |Par_9||4879||340ng/ulx 1 tube||6ul||14ul||130ul||62.1ng/ul x 10ul |- |Par_10||4526||380ng/ulx 1 tube||6ul||14ul||130ul||34.6ng/ul x 10ul |- |} *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 I got low concentration after bis-conversion. i quantified the gDNA concentration with nanodrop and redo the bis-conversion. {| 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''' |- |Par_1||5028||111.9ng/ul x 1 tube||19ul||1ul||130ul||90.8ng/ul x 10ul |- |Par_2||4735||150.5ng/ulx 1 tubes||14ul||6ul||130ul||86.7ng/ul x 10ul |- |Par_3||4789||118.7ng/ulx 1 tubes||19ul||1ul||130ul||74ng/ul x 10ul |- |Par_4||5171||143.9ng/ulx 1 tubes||14ul||6ul||130ul||98.3ng/ul x 10ul |- |Par_5||1818||113.5ng/ulx 1 tubes||19ul||1ul||130ul||69ng/ul x 10ul |- |Par_6||1947||151.4ng/ul x 1 tubes||14ul||6ul||130ul||109.7ng/ul x 10ul |- |Par_7||1741||133ng/ul x 1 tube||18ul||2ul||130ul||84.5ng/ul x 10ul |- |Par_8||4977||92.7ng/ulx 1 tube||20ul||0ul||130ul||73.4ng/ul x 10ul |- |Par_9||4879||190.6ng/ulx 1 tube||11ul||9ul||130ul||110.3ng/ul x 10ul |- |Par_10||4526||102.5ng/ulx 1 tube||20ul||0ul||130ul||83.1ng/ul x 10ul |- |} ===capture the samples with two cpg97k subsets=== The probe set was prepared by Alan. {| 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+cpg97k_A(25.6ng/ul_06/18) vol+suppress oligo+H2O''' | align="center" style="background:#f0f0f0;"|'''template+cpg97k_B(21ng/ul_06/18) vol+suppressor oligo+H2O''' |- |Par_1||5028 ||90.8ng/ul ||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- |Par_2||4735||86.7ng/ul ||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- |Par_3||4789||74ng/ul||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- |Par_4 ||5171||98.3ng/ul||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- |Par_5 ||1818||69ng/ul||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- |Par_6 ||1947||109.7ng/ul||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- |Par_7||1741||84.5ng/ul||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- |Par_8||4977||73.4ng/ul||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- |Par_9||4879||110.3ng/ul||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- |Par_10||4526||83.1ng/ul||1ul||4+2.5ul+1ul+1.5ul||4+2ul+1ul+2ul |- | ||positive control||Jurkat bis-gDNA 200ng/ul||1ul||1+1ul+1ul+6ul|| |- | ||negative control||0||1ul||0+1ul+1ul+6ul|| |} dNTP concentration 10mM(NEB) H2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul Stoffel; 0.5U/ul AmpLigase; 1mM dNTP: 0.5ul 0.1ul 0.1ul 0.2ul (10mM)0.1ul x100 50ul 10ul 10ul 20ul 10ul ===PCR=== Template 10ul x11 2X iProof Mastermix 50ul 550ul AmpF6.3SoL (10uM) 4ul 44ul AmpR6.3SoL (10uM) 4ul 44ul 50X SYBG I 0.4ul 4.4ul H2O 31.6ul 347.6ul 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 ->(98C 10S -> 72C 20S) x 8 ->72C 3 min -> 15C hold. [[Image:20090703_Parkinson_sample_cpg97k_A.jpg]]20090703_Parkinson_sample_cpg97k_A [[Image:20090703_Parkinson_sample_cpg97k_B.jpg]]20090703_Parkinson_sample_cpg97k_B I purified all samples with qiaquick column. Nanodrop quantification and mix the subset A and B captured targets with 1:1 ratio: ===shotgun library construction=== ====PCR amplification with AmpF6.3NH2/AmpR6.3NH2 (2009-07-14)==== I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample. reaction system x4 x10 H2O 45ul 180ul 2x Mastermix 50ul 200ul AmpF6.3NH2(10uM) 2ul 8ul AmpR6.3NH2(10uM) 2ul 8ul 50x SYBG I 0.4ul 1.6ul template 1ul/each for purified cpg97k Total 100ul 800ul 94C 2min -> (94C 45S -> 55C 45S -> 72C 45S) x 8 ->72C 3 min -> 15C hold. [[Image:20090714_Parkinson samples with AmpF6.3NH2 reamplification.jpg]]20090714_Parkinson samples with AmpF6.3NH2 reamplification I purified the samples with Qiaquick column. Quantify with nanodrop and send the samples to Billy. ====Sample Par. ship to Billy==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Concentration (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Total Amt. (ng)''' |- | Par_1||21.22||30||636.6 |- | Par_2||15.25||30||457.5 |- | Par_3||18.05||30||541.5 |- | Par_4||19.97||30||599.1 |- | Par_5||29.63||30||888.9 |- | Par_6||15.82||30||474.6 |- | Par_7||35.41||30||1062.3 |- | Par_8||46.59||30||1397.7 |- | Par_9||22.29||30||668.7 |- | Par_10||30.4||30||912 |- | |} ====endrepair with epicentre kit(2009-07-20)==== 100 ul DNA 15 ul 10X End-Repair Buffer 15 ul dNTP Mix 15 ul ATP 3 ul End-Repair Enzyme Mix 150 ul Total reaction volume Incubate at room temperature for 45 minutes. Purify with minelute column. ==== A tail addition==== x8 Blunt-ended DNA 10ul 80 10X Klenow buffer 1.5ul 12 1mM dATP 3ul 24 Klenow fragment (exo-) 1ul 8 37C 30min, purified with MinElute columns, eluted with 20ul EB. After A tail, I did size selection for No7 and positive control, then do the adaptor ligation. <BR>After adaptor ligation, i goes to PCR directly without size selection again. <BR>However, I can only see the positive control product only. So I skipped the size selection before adaptor ligation for No 1~6, and do the size selection after adaptor ligation. However, the expected band after PCR is so weak that I am not sure the targets were successfully amplified. ====adaptor ligation==== DNA 20ul 10x QuickLigase buffer (enzymatic) 3ul 20uM Adaptor oligo mix 3ul T4 DNA QuickLigase (enzymatic) 2ul Incubate at RT for 15 minutes. Purified with Qiaquick columns, eluted with 12ul EB ====PCR==== Template 15ul 2x iProof mix 50ul Solexa_PCR_up (10uM) 4ul Solexa_PCR_lo_PE (10uM) 4ul H2O 30ul 50X SYBG I 0.2ul [[Image:20090721_shotgun_libr_par7_positive control.jpg]]20090721_shotgun_libr_par7_positive control [[Image:20090721_shotgun_libr_par1~6.jpg]]20090721_shotgun_libr_par1~6 ===redo the shotgun library starting from PCR amplification with AmpF6.3NH2/AmpR6.3NH2 (2009-07-22)=== I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample. reaction system x4 x17 H2O 50ul 200ul 3400 2x Mastermix 50ul 200ul 3400 AmpF6.3NH2(100uM) 0.2ul 0.8ul 13.6 AmpR6.3NH2(100uM) 0.2ul 0.8ul 13.6 50x SYBG I 0.4ul 1.6ul 27.2 template 1ul/each for purified cpg97k Total 100ul 800ul 94C 2min -> (94C 45S -> 55C 45S -> 72C 45S) x 8 ->72C 3 min -> 15C hold. I did the PCR reamplification with Amp6.3 primers with Parkinson's sample 1 and 10 with the same PCR condition. ====Sample Par. ship to Covaris (2009-07-27)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Total Amt. (ng)''' |- | Par_1||580 |- | Par_2||650 |- | Par_3||580 |- | Par_4||520 |- | Par_5||640 |- | Par_6||500 |- | Par_7||490 |- | Par_8||840 |- | Par_9||590 |- | Par_10||400 |- | |} ====shotgun library construction 2009-08-05 refer to LabNotes on [http://genome-tech.ucsd.edu/LabNotes/index.php/Jie:LabNotes/CpgSeq/2009-8-5]====
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