Jie:LabNotes/Protocol/exon capture system
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Capture system for exon[edit]
the molar ratio of probe/target should be at least 125:1 or 250:1.
reaction system H2O 5ul 10x Ampligase buffer 1ul cosmic probe 2ul template (100ng/ul) 2ul
Reaction program:
95c 10min -> 63C 14h -> add 1ul SLN mix(0.2mM dNTP) -> 60C 4h ->
8 cycles (95C 1min -> 0.1C/sec to 60C, 60C for 4h), add SLN mix every 12 hours -> 95C 1min ->
when temperature cools down to 37C, add 2ul Exonuclease I/III mix immediately -> 37C 2h -> 94C 5min -> 4C hold.
SLN mix: 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
PCR[edit]
Template 13ul 2X iProof Mastermix 50ul AmpF6.2SoL (10uM) 4ul AmpR6.2SoL (10uM) 4ul 50X SYBG I 0.8ul H2O 28.2ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 13 -> 72C 5 min -> 15C hold.