Matt:LabNotes/2015-10-19

From ZhangLabWiki
Jump to navigation Jump to search

CA12k_Nov2014 Expansion PCR[edit]

Expansion PCR[edit]

  • Matt:LabNotes/2014-12-18 conclusion: 21 cycles of PCR starting with 100nM seed oligos
  • Divide into 2 Biorad low qPCR tubes with 67.5ul each
    • V4 primers - CA12k_Nov2014_V4
' Contig of Exons (V4)
CA12k_Nov2014 Seed Oligos (900nM) 15
2X KAPA SYBG MM 67.5
100uM AP1V4U 0.6
100uM AP2V4 0.6
H2O 51.3
Total 135

Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold

File:20151019 ExpansionPCR CA12kNov14V4.PNG

  • Purified each with 1 Qiagen column and eluted with 50ul H2O
  • Nanodrop:
    • V4: 5.3ng/ul -> 5.3ng/ul / (188bp*660Da/bp) = 42.7nM
  • Diluted to 10nM
    • V4: 100ul x 42.7nM = 427ul x 10nM
      • Add 327ul H2O

Update Jan 26 2016[edit]

  • Remeasured concentration of seed oligos due to low yields after 15 cycles of Production PCR
  • Erin took 10ul for Qubit dsDNA measurement
    • 168 ng/mL = ~1nM