AlanFung:LabNotes/Probe/2014-1-7

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GP1 V6 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_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  

PCR Profile[edit]

File:01072014 Expansion.JPG

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

qiaquick[edit]

  • one column for one reaction
  • 6x pb buffer
  • wash twice with PE Buffer
  • elute with 50ul EB Buffer

Nanodrop[edit]

File:01072014 nanodrop.JPG

  • 10.7ng/ul * 50ul = 535ng
  • 120.44nM
  • Stored in -20C (Alan Misc. #4)

Dilute probes to 10nM[edit]

  • Add 24.9ul probes to 275.1ul of EB buffer
  • Stored in -20C (Alan Misc. #3)

Production PCR (8 wells)[edit]

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[edit]

  • 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[edit]

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

Nanodrop[edit]

File:01072014 nanodrop 2.JPG

  • 82.7ng/ul*120ul ~9.9ug yield

Exonuclease digestion to generate SS-DNA[edit]

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[edit]

  • ssDNA: 37.6ng/ul * 80ul = 3ug

File:01082014 nanodrop.JPG

Removal of amplification adapters[edit]

  • 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[edit]

  • Elute with 40ul EB Buffer

Nanodrop[edit]

  • ssDNA: 43.8ng/ul *40ul ~1.75ug

File:01102014 nano.JPG

2D Gel Size Selection[edit]

  • I will use one 6% TBE-Urea gel 2D well to perform purification (one gel can handle 2-2.5ug)
  • Mix 40ul 2X TBE-Urea sample buffer in a 1:1 ration to make the final volume to 80ul
Components 1 lane
10bp DNA ladder 1 ul
2X TBE-Urea sample buffer 5ul
H2O 4ul
Total Volume 10ul
  • Pre-run 6%TBE –urea (TBU) gel at 250 volts for 10-15 min in 0.5X TBE buffer.
  • Heat DNA and DNA ladder-loading dye mixture at 75 C for 10mins, quickly transfer to ice, wait for 1 min
  • load gel with ladder and probes
  • Run gel at 200V for 30 mins
  • Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min
  • Prepare 3X gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube
  • Cut out the correct band chop the band into small slices, split into 3x shearing assembly.
  • Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
  • Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 50C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
  • In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm) and then transfer supernatant to 1.5mL tube.

File:ZhangLab 2 2014-01-10 15hr 20min.jpg

ETOH Precipitation[edit]

  • Add 0.1x of 3M sodium acetate and 2.5x 100% ethanol.
  • Add 1uL Glycoblue so that one can precipitate better and see the DNA.
  • Freeze in -70C overnight spin at 14,000 rpm for 15 min at 4C, discard supernatant with pipette.
  • Add 500 uL of 75% ethanol to wash DNA, spin at 8000 rpm for 5 min at 4C, discard supernatant with first large tip and then small tip to remove as much liquid as possible.
  • Let dry in hood ~ 10 min
  • Resuspend with 15ul ddh2O for each tube (45ul total)
  • Stored in (Maytag -20C Alan Misc. #2)

Quantify probes with qubit ssDNA because glycoblue will interfere with nanodrop[edit]

  • 5.88ng/ul *45ul = 264.6ng