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

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 90: Line 90:


|}
|}
==Realtime-PCR profile==
*95C 30sec
*(95C 10sec → 55C 45sec → 70C 45sec) x 15 cycles
*72C 3min
*Hold at 15C

Revision as of 00:36, 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

Components 1x rxn (ul) Final Conc
GP-1 (689nM) 14.51 100nM
KAPA SYBR FAST Master Mix (2X) 50 1X
100uM AP1V41U 0.4 400nM
100uM AP2V4 0.4 400nM
H2O 34.69  
Total 100  

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  

Realtime-PCR profile

  • 95C 30sec
  • (95C 10sec → 55C 45sec → 70C 45sec) x 15 cycles
  • 72C 3min
  • Hold at 15C