Revision as of 21:19, 8 April 2016 by >Mzcai
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
- 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
Reference protocol
- 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
|
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
Gel Size Selection
TBU Gel Check