AlanFung:LabNotes/Probe/2014-1-7: Difference between revisions

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==Nanodrop==
==Nanodrop==
[[File:01072014_nanodrop.JPG|400px]]
*10.7ng/ul * 50ul = 535ng  
*10.7ng/ul * 50ul = 535ng  
*120.44nM
*120.44nM
==Dilute probes to 10nM==
==Dilute probes to 10nM==
*Add 24.9ul probes to 275.1ul of EB buffer
*Add 24.9ul probes to 275.1ul of EB buffer

Revision as of 00:42, 9 January 2014

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  

Nanodrop

File:01072014 nanodrop.JPG

  • 10.7ng/ul * 50ul = 535ng
  • 120.44nM

Dilute probes to 10nM

  • Add 24.9ul probes to 275.1ul of EB buffer

Production PCR (8 wells)

Components 1x rxn (ul) 8x rxn (ul)
10nM 1st round amplicon 0.4 3.2
KAPA SYBR FAST MM (2X) 50 400
100uM AP1V61U 0.4 3.2
100uM AP2V6 0.4 3.2
H2O 48.8 390.4
Total 100 800

Realtime-PCR profile

  • QPCR done on ependorf
  • Left:GP1_V4 Right:GP1_V6
  • 95C 30sec
  • (95C 10sec → 55C 20sec → 72C 30sec) x 15 cycles
  • 72C 2min
  • Hold at 15C

File:01072014 Production.JPG

Qiaquick Column 4 Total

  • Pool 2 rxn into one column
  • Elute with 30ul EB buffer

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File:01072014 nanodrop 2.JPG

  • 82.7ng/ul*120ul ~9.9ug yield

Exonuclease digestion to generate SS-DNA

Components 1x rxn (ul)
GP1_V6 Purified amplicon (~10ug) 120
10X lambda Exonuclease Buffer 15
Lambda Exonuclease 6.7
H2O 8.3
Total 150
  • Incubate at 37 C for 1 hr on thermocycler
  • Purify with each set in 3x QIAquick columns as described above (may use 7X volume of buffer PB for ssDNA
  • Elute each column with 40 ul EB buffer (Total volume ~ 240 ul)

nanodrop

  • ssDNA: 37.6ng/ul * 80ul = 3ug

File:01082014 nanodrop.JPG

Removal of amplification adapters

  • In 200 ul PCR tube, add 5ul USER enzyme to 80ul probes
  • Incubate at 37 C for 3 hr on thermocycler.
  • Add 15 ul of the following components to the USER digested DNA tubes (10ul 10X DPNII Buffer, 5ul 100uM RE-DpnII-V6 guide oligo)
  • On thermocycler, incubated at 94 C for 2 min, cool down temperature to 37C for 3 min
  • Keep the tube on thermocycler.
  • Add 5 ul of DpnII and mix well by pipetting up and down
  • Incubate at 37 C for 2 hr.

Qiaquick Column Purification

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