Matt:LabNotes/2013-7-11: Difference between revisions

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*[http://genome-tech.ucsd.edu/LabNotes/index.php/Illumina_GA/Oligo_info#Primers_for_LC_Sciences_library-free_protocol Primers for Agi26k]
*[http://genome-tech.ucsd.edu/LabNotes/index.php/Illumina_GA/Oligo_info#Primers_for_LC_Sciences_library-free_protocol Primers for Agi26k]
*Primers for CES36k18bp: AmpF6.3NH2 and AmpR6.3NH2
*[http://genome-tech.ucsd.edu/LabNotes/index.php/Illumina_GA/Oligo_info#Library_Free_Multiplexing_Primers Primers for CES36k18bp: AmpF6.3Sol and AmpR6.3Ind48]


{| {{table}} border = 1
{| {{table}} border = 1
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| 3||cDNA-Agi26k_20gap||Indx78
| 3||cDNA-Agi26k_20gap||Indx78
|-
|-
| 4||gDNA-CES36k18bp||N/A
| 4||gDNA-CES36k18bp||Indx48
|} <br>
|} <br>


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   Program
   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
   *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 | 500px]]
*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

Latest revision as of 23:23, 11 July 2013

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