AlanFung:LabNotes/Probe/2013-12-11

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Revision as of 18:41, 12 December 2013 by >Alan6017518 (→‎Purify with one column per reaction)
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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

Qiaquick Column Purification

Purify with one column per reaction

  • In 1.5mL tube, add 6x volume of PB buffer to each tube with PCR product and mix (600ul to 100 ul of PCR product)
  • Transfer 700ul of mixture to each qiaquick column
  • spin at 14,000rpm for 1". Discard supernatant
  • Add 750ul wash buffer to each column
  • spin at 14,000rpm for 1". Discard supernatant
  • repeat wash with wash buffer
  • transfer column to 1.5mL lowbind tube
  • Add 50ul 55C EB buffer to each column and incubate in 37C for 5"
  • Elute DNA by spinning at 14,000 rpm for 1 min.
  • Pool the purified amplicon into the same tube (total volume ~100ul).
  • Measure DNA concentration by Nanadrop and calculate the total yield (dsDNA).
  • Dilute purified 1st round amplicon to 10nM with TE buffer.