AlanFung:LabNotes/Probe/2014-1-7

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Revision as of 19:45, 7 January 2014 by >Alan6017518 (→‎Expansion PCR)
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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

GP1_V6 Expansion PCR

Components 1x rxn (ul) Final Conc
GP-1 (689nM) 14.51 100nM
KAPA SYBR FAST Master Mix (2X) 50 1X
100uM pAP1V61U 0.4 400nM
100uM AP2V6 0.4 400nM
H2O 34.69  
Total 100