Jie:LabNotes/ExonomeSeq/2009-1-19
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capture of Jurkat with long cosmic probe and ds short cosmic probe[edit]
tube 1: short cosmic3760 (19.3ng/ul,4/29/08. requantify today: 12ng/ul) tube 2: long cosmic 3760 (14ng/ul) tube 3: long cosmic 3760 (14ng/ul) tube 4: ds short comic 3760 (10ng/ul) tube 5: ds short cosmic 3760 (10ng/ul) tube 6: negative control for short probe tube 7: negative control for long probe tube 8: negative control for ds short probe
Capture reaction:
reaction system 1 2 3 4 5 6 7 8 H2O 6 6.2 4.4 4 2 7 7.2 5 10x Ampligase Buffer 1 each probe 2 1.8 3.6 4 6 2 1.8 4 Jurkat gDNA(200ng/ul) 1 1 1 1 1 0 0 0
Reaction program:
95c 10min -> 63C 14h -> add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 60C 4h ->
12 cycels (95C 1min -> 0.1C/sec to 60C, 60C for 4h 55C 1h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 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]
x8 Template 10ul 2X iProof Mastermix 50ul 400ul AmpF6.2SoL (10uM) 2ul 16ul AmpR6.2SoL (10uM) 2ul 16ul 50X SYBG I 0.4ul 3.2ul H2O 35.6ul 284.8ul
98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 13 -> 72C 5 min -> 15C hold.
Short Cosmic 3760 had only 17cycles while long probe had 21 cycles.
File:20090122 long and ds cosmic capture.jpglong and ds cosmic capture
Qiaquick column purification: short probe: 15.3ng/ul (36nM) x 30ul; long probe (low con.): 32.9ng/ul; long probe (high con.): 18.7ng/ul (30nM) x 30ul;
Sent 15ul short probe captured amplicon (36nM) and long probe (high con.) captured amplicon (30nM) to Yuan Gao.