Alice:LabNotes/2008-11-24

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Extend Cosmic Probe[edit]

Spacer insertion

template (1) 860nM                       1.5 ul 
spacer mix (100nM each)                   20 ul
10x Ampligase Buffer                       5 ul
Ampligase(5U/ul)                           1 ul
ddH2O                                   15.6 ul
----------------------------------------------------------
total                                     50 ul

95C 5min -> 65C 40min

Add 2ul of ExoI&III 37C for 2hrs -> 94C 10min
PCR amplification after spacer insertion
                                                   system 1     system 2
H2O                                                   35ul        40
SYBG                                                 0.6ul       0.6
primer mix (AP1V4IU+AP2V4)                           0.2ul       0.2
Taq master mix                                        50ul        50
Template (from previous step)                         15ul        10
----------------------------------------------------------------------
total                                                100ul      100ul
94C 2min -> 94C 45sec -> 58C 45sec -> 72C 30 sec ->plate read -> 20 cycle -> 72C 3min -> 15C

TBE gel result:
File:ZhangLab 2 2008-11-26 10hr 18min-small.jpg
lane 1: PCR result of reaction system 1
lane 2: PCR result of reaction system 2