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Jie:LabNotes/CpgSeq/2009-5-20
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==set up the capture reaction== {| 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+cpg92k+H2O''' | align="center" style="background:#f0f0f0;"|'''template+control probes+H2O''' | align="center" style="background:#f0f0f0;"|'''reaction condition''' |- |1||bis_Jurkat||255.6ng/ul||1ul||1+4ul(#1_22.3ng/ul_05/20)+4ul||||55C anneal |- |2||bis_Jurkat||255.6ng/ul||1ul||1+4ul(#1_22.3ng/ul_05/20)+4ul||||60C anneal |- |3||bis_Jurkat||255.6ng/ul||1ul||1+3ul(#2_17ng/ul_05/20)+5ul||||55C anneal |- |4||bis_Jurkat||255.6ng/ul||1ul||||1+1ul(cpg30k,50ng/ul,06/13/08)+7ul||55C anneal |- |5||Jurkat||142.8ng/ul||1ul||||1+1ul(cosmic,19.3ng/ul,04/29/08)+7ul||55C anneal |- |6||09_1||95.4ng/ul||1ul||2+5ul(#2_17ng/ul_05/20)+2ul||||55C anneal |- |7||09_7||311.2ng/ul ||1ul||2+5ul(#2_17ng/ul_05/20)+2ul||||55C anneal |- |8||negative control||0||1ul||0+4ul(#1_22.3ng/ul_05/20)+5ul||||55C anneal |} dNTP concentration 10mM(NEB) ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP: 0.5ul 0.1ul 0.1ul 0.2ul (10mM)0.1ul x30 15ul 3ul 3ul 6ul 3ul 95c 10min -> 55C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 55C 4h -> <BR>4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle-> 95C 5min -> <BR>add 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold. tube2: 95c 10min -> 60C 24h ->add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 1mM dNTP) -> 60C 4h -> <BR>4 cycels (95C 1min -> 55C for 4h),add 2ul more SLN mix after 1st cycle -> 95C 5min -> <BR>add 2ul Exonuclease I/III mixadd 2ul Exonuclease I/III mix as soon as the temperature is lowered to 37C -> 37C 1h -> 94C 5min -> 4C hold.
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