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
- 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
- Make 2X B&W (Binding and Wash) Buffer
- 16ml 5uM NaCl + 80ul 5uM EDTA + 100ul 4uM Tris-HCl + 23.82ml H2O
- Resuspend beads by vortexing
- Transfer 100ul of beads (10ug/ul) to new tube
- Pull down by magnet 2min and remove supernatant
- Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant
- Resuspend in 200ul 2X B&W Buffer
- Add 200ul of 2.5uM PKP2_Control
- Incubate 15min at RT gently rotating
- Pull down with magnet
- Wash 3x with 1X B&W Buffer
- 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
- Use CA12kNov2014_V4 Probes to Capture PKP2_Control oligos attached to streptavidin beads to test
Sample Groups
- PKP2_Control on beads
- 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 |