Capture Test of CES36k18bp: Difference between revisions

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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
 


The Probe:Target ratio is 100:1
The Probe:Target ratio is 100:1
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Probe:
Probe:
  CES36k18bp: 5.6ng/ul;36,000 probes; 170nM
  CES36k18bp: 5.6ng/ul; 36,000 probes; 170nM
  Volume of CES36k18bp needed = (200ng/3pg * 100 * 36000)/(6.0 * 10^23 * 170 nM) = 2.35ul
  Volume of CES36k18bp needed = (200ng/3pg * 100 * 36000)/(6.0 * 10^23 * 170 nM) = 2.35ul


'''Reaction Setup'''
'''Reaction Setup'''
Capture Reaction:
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==

Revision as of 23:40, 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 Capture Reaction:

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