Matt:LabNotes/2013-7-11

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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-7-9

Amplification with Sequencing Adapters[edit]

Tube # Sample Index#
1 NTC-Agi26k_20gap Indx7
2 gDNA-Agi26k_20gap Indx77
3 cDNA-Agi26k_20gap Indx78
4 gDNA-CES36k18bp Indx48


Components 1X Reaction 5X Reaction
Captured Template 1 0
10uM Forward + Indx 0.4 0
10uM Reverse 0.4 2.0
2X KAPA SYBG FAST MM 12.5 62.5
H2O 10.7 53.5
Total 25 125
 Program
 *98°C 30sec -> (98°C 10sec -> 52°C 30sec -> 72°C 30sec) x8 cycles -> (98°C 10sec -> 72°C 30sec) x16 cycles -> 72°C 3 min -> 15°C hold

File:071113 PCRAgi20gapProbes PosCtrl.JPG

  • Expected curves from NTC and PosCtrl (CES36k18bp probes)
  • Means the low efficiency of the Agi26k_20gap probes in previous capture reaction was not due to experimental error
    • The NTC from previous reaction was most likely due to not adding ExoI/III mix and/or contamination