Matt:LabNotes/2016-3-31

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Bead with PKP2 Target for DARTFISH Positive Control

  • Using PA gel may prevent diffusion of probes and enzymes during DARTFISH
  • To test this and to have a positive control for future experiments need to design a synthetic target
    • Attach target to magnetic streptavidin bead (Dynabeads MyOne C1)
  • Design oligonucleotide with biotin 5' modification
    • Chose PKP2 gene because haven't detected it in any DARTFISH samples
    • Also this specific padlock probe has very high efficiency when measured in tube
 PKP2_control	/5BiosG/AAAAAAGAGATGGCTGTCTTTTTCACACTTGGGTCACCAACATGCAGCATCTTTC

Link PKP2_control oligo to Streptavidin Bead

  1. Make 2X B&W (Binding and Wash) Buffer
    • 16ml 5uM NaCl + 80ul 5uM EDTA + 100ul 4uM Tris-HCl + 23.82ml H2O
  2. Resuspend beads by vortexing
  3. Transfer 100ul of beads (10ug/ul) to new tube
  4. Pull down by magnet 2min and remove supernatant
  5. Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant
  6. Resuspend in 200ul 2X B&W Buffer
  7. Add 200ul of 2.5uM PKP2_Control
  8. Incubate 15min at RT gently rotating
  9. Pull down with magnet
  10. Wash 3x with 1X B&W Buffer
  11. Resuspend in 1ml 1X PBS

Measure DNA Concentration

  • Use Qubit ssDNA kit to measure amount of ssDNA on beads
    • Assume free biotin-oligos have been all washed away
    • Measure only streptavidin bead as well for base-line
  • Beads + DNA: 1.56ng/ul
  • Beads only: 164pg/ul
  • Don't trust the quantitation but it shows ssDNA was bound to beads

Capture with CA12kNov2014 V4 in tube

Reference protocol

  • Use CA12kNov2014_V4 Probes to Capture PKP2_Control oligos attached to streptavidin beads to test

Sample Groups

  1. PKP2_Control on beads
  2. Beads only (NTC)
Sample # Sample Description Probes (ul) Target (ul) Target Conc (ng/ul) 10X Ampligase Buffer H2O Total
1 PKP2_Control 3.5 1 1.56 3 22.5 30
2 NTC 3.5 1 0 3 22.5 30
  • Add 40ul Mineral Oil on top

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
    • In BioRad Thermalcycler program says: -0.2C per cycle every 30sec
  • -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
  • -> 55 C 20h (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

AmpLigase enzyme mix

Components Stock conc. Unit Final conc. Unit Prepare volume 30ul
AmpLigase 5 U/ul 0.5 U/ul 2.00
10x AmpLigase Buffer 10 x 1 x 2.00
H2O 16.00
Total 20.00

PCR + Sequencing Adapters

Primers

Primer Sequence Index #
ISB_CA_AF AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG
ISB_CA_AR.T1 CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG Indx1
ISB_CA_AR.T2 CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG Indx2
ISB_CA_AR.T3 CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG Indx3
Sample Index Forward Primer Reverse Primer
PKPK2_Control 3 ISB_CA_AF ISB_CA_AR.T3
NTC 1 ISB_CA_AF ISB_CA_AR.T1

PCR Test

Components 1X Volume
Captured template 10
10uM Forward Primer 0.4
10uM Reverse Primer 0.4
2X KAPA SYBG MM 12.5
H2O 1.7
Total 25
  • Used 10ul of template instead of 1ul because expect low amount due to only target for 1 of the 3,500 padlock probes
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x25

File:2016-04-04 PadlockProbe PKP2 in tube Test Sequence Adapter PCR.JPG

PCR

Components 1X Volume
Captured template 12
10uM Forward Primer 2
10uM Reverse Primer 2
2X KAPA SYBG MM 50
H2O 34
Total 100
  • Split into duplicates of 50ul because Biorad cycler can't handle >50ul reaction volume
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

File:2016-04-05 PadlockProbe PKP2 in tube Sequence Adapter PCR.JPG

  • Bead purification with 1.4:1 Beads to amplicon volume ratio
    • Eluted with 50ul total for each sample

TBU Gel Check

  • Load 2ul of each sample + 2ul loading dye

File:20160406 PP capture seq library.jpg

  • Lane 1: Low Mass Ladder
  • Lane 2: Erin's capture of gDNA from CA12kNov2014_V4 that had 3 extra cycles during Production PCR
  • Lane 3: Erin's capture of NTC from CA12kNov2014_V4 that had 3 extra cycles during Production PCR
  • Lane 4: PKP2_Control captured

Gel Size Selection

TBU Gel Check