AlanFung:LabNotes/Probe/2014-1-7

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Revision as of 19:20, 7 January 2014 by >Alan6017518 (Created page with "==GP1 V6 Padlock Probe Production== ==Overview== *Expansion PCR *Production PCR *Exonuclease digestion to generate SS-DNA *Removal of amplification adapters *Probe purificati...")
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GP1 V6 Padlock Probe Production

Overview

  • Expansion PCR
  • Production PCR
  • Exonuclease digestion to generate SS-DNA
  • Removal of amplification adapters
  • Probe purification by PAGE size-selection

Expansion PCR

  • Probe concentration from CustomArray 32.71ng/ul volume 80uL Amount 2.62ug
  • Probe size 146bp

Convert ng/ul to nM

  • Molar Mass of ssDNA probe: 146bp * (325Da/bp) = 146bp * (325 g/mol/bp) = 47450 g/mol
  • (32.71ng/ul * 1*10^6 ul/L) / (47450g/mol) = 689.36nM
  • I will use 100nM final concentration for the expansion PCR

Expansion PCR

Components 1x rxn (ul)
GP-1 (689nM) 14.51
KAPA SYBR FAST MM (2X) 50
100uM AP1V41U 0.4
100uM AP2V$ 0.4
H2O 34.69
Total 100