Capture Test of CES36k18bp: Difference between revisions

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'''Probe and Sample'''
'''Probe and Sample'''


Probes: CES36k18bp, 5.6ng/ul
Probes: CES36k18bp, 5.6ng/ul
Sample is Jurkat DNA standard, 100ng/ul (2.00ul)
Sample is Jurkat DNA standard, 100ng/ul (2.00ul)
 
The Probe:Target ratio is 100:1
 
The Probe:Target ratio is 100:1


'''Calculations'''
'''Calculations'''
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'''Reaction Setup'''
'''Reaction Setup'''
Capture Reaction:
 
  Jurkat DNA:          2.00ul
  Jurkat DNA:          2.00ul
  10X AMPLigase Buffer: 1.50ul
  10X AMPLigase Buffer: 1.50ul

Revision as of 23:41, 5 January 2010

Capture Protocol

Probe and Sample

Probes: CES36k18bp, 5.6ng/ul
Sample is Jurkat DNA standard, 100ng/ul (2.00ul)
The Probe:Target ratio is 100:1

Calculations

Probe:

CES36k18bp: 5.6ng/ul; 36,000 probes; 170nM
Volume of CES36k18bp needed = (200ng/3pg * 100 * 36000)/(6.0 * 10^23 * 170 nM) = 2.35ul

Reaction Setup

Jurkat DNA:           2.00ul
10X AMPLigase Buffer: 1.50ul
CES36k18bp:           2.35ul
H2O:                 14.15ul

94C, 5min -> -0.1C/sec to 60C -> 60C, 24h -> add 2ul SLN mix -> 60C, 24h -> 95C, 2min -> 37C, 1min -> add 2ul Exo I&III -> 37C, 2h -> 94C, 5min -> 4C hold

PCR

Template:           8ul
2X Phusion mix:    50ul
100uM AmpF6.3NH2: 0.2ul
100uM AmpR6.3NH2: 0.2ul
50X SYBG:         0.4ul
H2O:               30ul

Results