Matt:LabNotes/2015-3-25

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Revision as of 20:47, 26 March 2015 by >Mzcai
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CA12k_Nov2014_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 20
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4900
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:032515 CA12kNov14 ProductionPCR V4.JPG

Zymo DNA Clean & Concentrator 100

  • Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
    • Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
  • Elute with 540ul Elution buffer
    • Should elute with < 400ul next time because it won't all fit in column
      • Lost about 80ul and ended with low yield (50ug normally after EtOH precipitation and Qia purification)
  • Measured concentration with Nanodrop:

~460ul of V4 probes: 71.3 ng/ul => 32.8ug

Repeat Production PCR

  • 10nM 1st round amplicons
  • V4 master mix made in 1 15ml centrifugal tube
  • Poured into trough/reservoir and used multi-channel pipette to save time pipetting into pcr tubes
Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 20
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4900
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:032515 CA12kNov14 ProductionPCR V4.JPG

Zymo DNA Clean & Concentrator 100

  • Tried using reservoir & multi-channel pipette removing product from 6 strips of pcr tubes but spilled ~1ml (product+binding buffer) pouring from reservoir into 50ml tubes
  • Purify 10ml of PCR product with 1 Zymo-spin V Column/Reservoir
    • Add 50ml DNA Binding Buffer to 10ml product (Use 2 50ml tubes)
  • Elute with 400ul H2O
    • Somehow final volume is greater than 400ul
  • Measured concentration with Nanodrop:

~410ul of V4 probes: 76.1 ng/ul => 31.2ug

Lambda Exonuclease Digestion

  • Divide into 9 pcr tubes of ~110ul with total amplicon of <10ug each
Components Volume
Amplicon 96
10X Lambda Exo Buffer 12
Lambda Exonuclease 12
Total 120
  • Incubated at 37C for 1hr
  • Purified with 9 Zymo ssDNA/RNA columns
  • Eluted with 30ul each
  • Recombined and measured ssDNA with Nanodrop:
    • ng/ul x 270ul = ug (% yield)