AlanFung:LabNotes/Probe/2013-12-11

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GP1 Padlock Probe Production[edit]

Overview[edit]

  • Expansion PCR
  • Production PCR
  • Exonuclease digestion to generate SS-DNA
  • Removal of amplification adapters
  • Probe purification by PAGE size-selection

Expansion PCR[edit]

  • 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[edit]

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[edit]

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[edit]

File:12132013 GP1 V4.JPG File:12132013 GP1 V6.JPG

  • Left:GP1_V4 Right:GP1_V6
  • 95C 30sec
  • (95C 10sec → 55C 45sec → 70C 45sec) x 15 cycles
  • 72C 3min
  • Hold at 15C

Qiaquick Column Purification[edit]

Purify with one column per reaction[edit]

  • 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.

Nanodrop[edit]

GP1_V4 GP1_V6

  • Left:GP1_V4 Right:GP1_V6
  • GP1_V4:10ng/ul, 112.56nM
  • GP1_V6:9.9ng/ul, 111.44nM
  • Keep 1ul for Gel quantification

Dilute probes to 10nM[edit]

  • GP1_V4: Add 0.85ul 112.56nM probes to 8.75ul EBuffer
  • GP1_V6: Add 0.86ul 111.44nM probes to 8.74ul EBuffer

Production PCR (24 wells)[edit]

Mix the reaction mix in 50-mL tube Mix well and aliquot 100ul of reaction mix into each well with multichannel pipettes.

GP1_V4 Production PCR[edit]

Components 1x rxn (ul) 24x rxn (ul)
10nM 1st round amplicon 0.4 9.6
KAPA SYBR FAST Master Mix (2X) 50 1200
100uM AP1V41U 0.4 9.6
100uM AP2V4 0.4 9.6
H2O 48.8 1171.2
Total 100 2400

GP1_V6 Production PCR[edit]

Components 1x rxn (ul) 24x rxn (ul)
1st round amplicon 0.4 9.6
KAPA SYBR FAST Master Mix (2X) 50 1200
100uM pAP1V61U 0.4 9.6
100uM AP2V6 0.4 9.6
H2O 48.8 1171.2
Total 100 2400

Realtime-PCR profile[edit]

File:12132013 GP1 V4 production.JPG File:12132013 GP1 V6 production.JPG

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

EtOH precipitation[edit]

  • Transfer the amplified amplicon from 96-well plate into 50-mL disposable basins (Total volume ~9.6mL)
  • Aliquot 1.2 mL of amplified amplicon into 15-mL tube (4 tubes)
  • In each tube, add 4 ul of Glycoblue, 120 ul of 3M NaOAc, and 3 mL of 100 % EtOH (2.5 volume of 100% EtOH, 1/300 volume of Glycoblue, 0.1 volume of 3M NaOAc pH 5.2 or 5.5)
  • Mix well and leave at -80C for at least 30 min.
  • Precipitate by centrifugation at ~10,000xg for 30 min, at 4 C
  • Discard spnt and add 800ul of cold 75% EtOH to each tube.
  • Use 1 mL clean pipette tip transfer DNA pellet to 1.5 mL tube.
  • Rinse the pellet and spin the tube at 14,000 rpm for 5 min.
  • Discard spnt and air-dry the pellet for 3-5 min in the hood (PCR work station).
  • Resuspend DNA with 150 ul ddH2O mix well by pipetting up and down.

Qiaquick Re-purification[edit]

  • Add 6x volume of PB buffer to each tube (900ul to 150 ul of DNA)
  • Transfer 700 ul of the mixture to each QIAquick column (6 columns total, 3 for each set)
  • Spin at 14,000 rpm for 1 min. Discard the spnt.
  • Add 750 ul of PE buffer to each tube.
  • Spin at 14,000 rpm for 1 min. Discard spnt (repeat washing step with buffer PE)
  • Spin the empty column at 14,000 rpm for 1 min.
  • Transfer the column to a fresh 1.5 mL tube.
  • Add 50 ul of 55C EB buffer to each tube
  • Elute the DNA by spinning at 14,000 rpm for 1 min.
  • Pool the purified amplicon into the same tube (total volume ~100 ul for each set).
  • Measure DNA concentration by Nanadrop and calculate the total yield (ds-DNA).

Nanodrop[edit]

File:12142013 GP1 V4 ND.JPG File:12142013 GP1 V6 ND.JPG

  • Left: GP1_V4 Right GP1_V6
  • GP1_V4: 18.1ng/ul *150ul = 2715 ng
  • GP1_V6: 20.6ng/ul *150ul = 3090 ng

Exonuclease digestion to generate SS-DNA[edit]

Components 1x rxn (ul) 2x rxn (ul)
GP1_V4 Purified amplicon (~ 15ug) 75 150
10X Lambda Exonuclease Buffer 15 30
Lambda Exonuclease 10 20
H2O 50 100
Total 150 300
     
Components 1x rxn (ul) 2x rxn (ul)
GP1_V6 Purified amplicon (~ 15ug) 75 150
10X Lambda Exonuclease Buffer 15 30
Lambda Exonuclease 10 20
H2O 50 100
Total 150 300
  • 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[edit]

File:12142013 GP1 V4 ND SS.JPG File:12142013 GP1 V6 ND SS.JPG

  • Left: GP1_V4_SS Right: GP1_V6_SS
  • GP1_V4_SS: 2.3 ng/ul *120ul = 276ng
  • GP1_V6_SS: 3.3 ng/ul *120ul = 396ng
  • Yield is too low, may be too much lambda exo for the reaction and lead to over digestion?
  • Store probes in -20C for storage and troubleshoot

Removal of amplification adapters[edit]

  • SETUP RXN
  • Incubate at 37 C for 3 hr on thermocycler.
  • Add 15 ul of the following components to the USER digested DNA tubes
  • 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 Purification[edit]

  • Purify USER/DpnII digested amplicon with 4-6 QIAquick columns as described previously (use 7X volume of buffer PB).

Nanodrop ssDNA[edit]

  • Measure DNA concentration (ssDNA) by Nanodrop and calculate total yield.

Probe purification by PAGE size-selection[edit]

EtOH Precipitation[edit]

Qubit ssDNA[edit]