Capture Test of CES36k18bp: Difference between revisions
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'''Probe and Sample''' | '''Probe and Sample''' | ||
Probes: CES36k18bp, 5.6ng/ul | |||
Sample is Jurkat DNA standard, 100ng/ul | Sample is Jurkat DNA standard, 100ng/ul (2.00ul) | ||
The Probe:Target ratio is 100:1 | |||
SLN Mix: | |||
Stoffel 2U/ul + AMPLigase 4U/ul + dNTP 0.25mM | |||
'''Calculations''' | '''Calculations''' | ||
Probe: | Probe: | ||
CES36k18bp: 5.6ng/ul;36,000 probes; | 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 | |||
98C, 30sec -> (98C, 10sec -> 58C, 20sec -> 72C, 20sec -> read) x 20 -> 72C, 3min -> 4C hold | |||
==Results== | |||
The post-capture PCR was allowed to run to completion. The product from this PCR, 100ul total, was then run through a TBE gel to check for viable product. | |||
'''Gel Setup''' | |||
25bp Ladder: 0.5ul Ladder + 3ul 1X TE Solution + 3ul 6X Loading Dye | |||
Low Mass Ladder: 0.5ul Ladder + 3ul 1X TE Solution + 3ul 6X Loading Dye | |||
Sample (x2): 5ul Sample (PCR Product) + 5ul 6X Loading Dye | |||
The TBE gel was run at 250 volts for 20 min: | |||
[[image: CES_Capture_Test_Annotated.jpg]] |
Latest revision as of 00:57, 12 January 2010
Capture Protocol[edit]
Probe and Sample
Probes: CES36k18bp, 5.6ng/ul Sample is Jurkat DNA standard, 100ng/ul (2.00ul) The Probe:Target ratio is 100:1
SLN Mix: Stoffel 2U/ul + AMPLigase 4U/ul + dNTP 0.25mM
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
98C, 30sec -> (98C, 10sec -> 58C, 20sec -> 72C, 20sec -> read) x 20 -> 72C, 3min -> 4C hold
Results[edit]
The post-capture PCR was allowed to run to completion. The product from this PCR, 100ul total, was then run through a TBE gel to check for viable product.
Gel Setup
25bp Ladder: 0.5ul Ladder + 3ul 1X TE Solution + 3ul 6X Loading Dye Low Mass Ladder: 0.5ul Ladder + 3ul 1X TE Solution + 3ul 6X Loading Dye Sample (x2): 5ul Sample (PCR Product) + 5ul 6X Loading Dye
The TBE gel was run at 250 volts for 20 min: