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Jie:LabNotes/CpgSeq/2008-8-15
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==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.
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