Capture Test of CES36k18bp

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Revision as of 00:52, 12 January 2010 by >Jbrubake (→‎Results)
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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
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

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: File:CES Test.jpg