Matt:LabNotes/2016-3-31: Difference between revisions
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=Bead with PKP2 Target for DARTFISH Positive Control | =Bead with PKP2 Target for DARTFISH Positive Control= | ||
*Using PA gel may prevent diffusion of probes and enzymes during DARTFISH | *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 | *To test this and to have a positive control for future experiments need to design a synthetic target | ||
**Attach target to magnetic streptavidin bead | **Attach target to magnetic streptavidin bead (Dynabeads MyOne C1) | ||
*Design oligonucleotide with biotin 5' modification | *Design oligonucleotide with biotin 5' modification | ||
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==Link PKP2_control oligo to Streptavidin Bead== | ==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== | ==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== | ==Capture with CA12kNov2014 V4 in tube== | ||
[[Matt:LabNotes/2015-5-18 | 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) | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample #''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Probes (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Target (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Target Conc (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''10X Ampligase Buffer''' | |||
| align="center" style="background:#f0f0f0;"|'''H2O''' | |||
| align="center" style="background:#f0f0f0;"|'''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'''<br> | |||
* 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==== | |||
{| class="wikitable" style="text-align:center;{{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Stock conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''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== | ==PCR + Sequencing Adapters== | ||
====Primers==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Primer''' | |||
| align="center" style="background:#f0f0f0;"|'''Sequence''' | |||
| align="center" style="background:#f0f0f0;"|'''Index #''' | |||
|- | |||
| ISB_CA_AF||AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG|| | |||
|- | |||
| ISB_CA_AR.T1||CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG||Indx1 | |||
|- | |||
| ISB_CA_AR.T2||CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG||Indx2 | |||
|- | |||
| ISB_CA_AR.T3||CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG||Indx3 | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Index''' | |||
| align="center" style="background:#f0f0f0;"|'''Forward Primer''' | |||
| align="center" style="background:#f0f0f0;"|'''Reverse Primer''' | |||
|- | |||
| PKPK2_Control||3||ISB_CA_AF||ISB_CA_AR.T3 | |||
|- | |||
| NTC||1||ISB_CA_AF||ISB_CA_AR.T1 | |||
|} | |||
====PCR Test==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''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| 450px]] | |||
====PCR==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''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|450px]] | |||
*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|150px]] | |||
*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=== |
Latest revision as of 18:46, 15 April 2016
Bead with PKP2 Target for DARTFISH Positive Control[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- Use CA12kNov2014_V4 Probes to Capture PKP2_Control oligos attached to streptavidin beads to test
Sample Groups[edit]
- 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[edit]
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[edit]
Primers[edit]
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[edit]
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[edit]
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[edit]
- 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