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
Line 24: Line 24:


==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]]
[[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]

  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[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]

Reference protocol

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

Sample Groups[edit]

  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[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

Gel Size Selection[edit]

TBU Gel Check[edit]