AlanFung:LabNotes/Probe/2013-12-11: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 12: Line 12:
*Molar Mass of ssDNA probe: 146bp * (325Da/bp) = 146bp * (325 g/mol/bp) = 47450 g/mol
*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
*(32.71ng/ul * 1*10^6 ul/L) / (47450g/mol) = 689.36nM
 
*I will use 100nM final concentration for the expansion PCR
 
 
===GP1_V4 Expansion PCR===  
===GP1_V4 Expansion PCR===  
===GP1_V6 Expansion PCR===
===GP1_V6 Expansion PCR===

Revision as of 00:09, 12 December 2013

GP1 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_V4 Expansion PCR

GP1_V6 Expansion PCR