Matt:LabNotes/2015-1-26: Difference between revisions

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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold
<!--*Stopped after 15 cycles
*Stopped after 15 cycles<!--
[[File:010514_CA12kNov14_ProductionPCR_V4V7.JPG | 800px]]
[[File:010514_CA12kNov14_ProductionPCR_V4V7.JPG | 800px]]


===EtOH Precipitation===
===EtOH Precipitation===
*6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7
*6 5-ml tubes (with 8 wells of PCR product each) for V6 and 6 tubes for V8
**800ul PCR product
**800ul PCR product
**2000ul 100% EtOH
**2000ul 100% EtOH
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**80ul 3M NaOAc pH 5.2-5.5
**80ul 3M NaOAc pH 5.2-5.5


*Vortexed and put in -80C for overnight
*Vortexed and put in -80C for 1hr
*Centrifuged at 3000rpm at 4C for 30 min
*Centrifuged at 3000rpm at 4C for 30 min
*Discarded supernatant and added 800ul of cold 80% EtOH
*Discarded supernatant and added 800ul of cold 80% EtOH

Revision as of 22:57, 27 January 2015

CA12k_Nov2014 RevComp Probe Production

Expansion PCR

  • Matt:LabNotes/2014-12-18 conclusion: 21 cycles of PCR starting with 100nM seed oligos
  • Make 2 tubes:
    • V6 primers - CA12k_Nov2014_Contig_RevComp
    • V8 primers - CA12k_Nov2014_Const_RevComp
' Contig of Exons RevComp(V6) Constitutive Exons RevComp(V8)
CA12k_Nov2014 Seed Oligos (900nM) 15 15
2X KAPA SYBG MM 67.5 67.5
100uM AP1V6U 0.6 0
100uM AP2V6 0.6 0
100uM AP1V8U 0 0.6
100uM AP2V8 0 0.6
H2O 51.3 51.3
Total 135 135

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

File:012614 CA12kNov14 ExpansionPCR V6V8.JPG

  • Did not think V6 amplified correctly (possibly due to underpipetting a primer) but V4 showed a similar curve
    • Nanodrop also shows V6 amplified as much or more than V4
  • Purified each with 1 Qiagen column and eluted with 50ul H2O
  • Nanodrop:
    • V6: 11.8 ng/ul -> 11.8 ng/ul / (188bp*660Da/bp) = 95nM
    • V8: 16.2 ng/ul -> 16.2 ng/ul / (190bp*660Da/bp) = 129nM
  • Diluted to 10nM
    • V6: 48ul x 95nM = 456ul x 10nM
      • Add 408ul H2O
    • V8: 48ul x 129nM = 619ul x 10nM
      • Add 571ul H2O

Gel Check

  • Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye
  • Use 2ul 10nM Agi26k0gap expansion PCR'd probes as positive control

File:2015-01-26 1stRndAmplicon V6V8 Gel.jpg

  • Apparently Nanodrop is not accurate enough at low conc
  • V6 is missing a band at 188bp...
    • Try Expansion PCR with V6 primers on the last of the seed oligos

V6 Expansion PCR Try 2

Components Volume
CA12k_Nov2014 Seed Oligos (900nM) 14
2X KAPA SYBG MM 50
100uM AP1V6U 1
100uM AP2V6 1
H2O 34
Total 100

Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold File:012614 CA12kNov14 ExpansionPCR V6 try2.JPG

  • Stopped after 22 cycles
  • Purified each with 1 Qiagen column and eluted with 50ul H2O
  • Nanodrop:
    • V6: 17.9 ng/ul -> 17.9 ng/ul / (188bp*660Da/bp) = 144nM
  • Diluted to 10nM
    • V6: 48ul x 144nM = 691ul x 10nM
      • Add 643ul H2O

Production PCR

  • V6 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V6 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V6U (100uM) 0.4 20
AP2V6 (100uM) 0.4 20
H2O 49 2450
Total 100 5000
  • V8 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V8 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V8U (100uM) 0.4 20
AP2V8 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold

  • Stopped after 15 cycles