Matt:LabNotes/2016-8-21: Difference between revisions

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**For DNA template positive control only do 1 sample using 300ng gDNA
**For DNA template positive control only do 1 sample using 300ng gDNA


*Sample 1: gDNA template captured by V6 padlock probes with Ampligase at 95C->55C
*Sample 1: gDNA template captured by V6 padlock probes with Ampligase at 55C
**Standard padlock probe capture
**Standard padlock probe capture except keep at 55C whole time
*Sample 2: RNA template captured by V6 padlock probes with SplintR at 37C
*Sample 2: RNA template captured by V6 padlock probes with SplintR at 25C
**Should be ligated but non-specific because low temp
**Should be ligated but non-specific because low temp
*Sample 3: biotinylated and streptavidin bead linked RNA captured by V6 padlock probes with SplintR at 37C
*Sample 3: biotinylated and streptavidin bead linked RNA captured by V6 padlock probes with SplintR at 25C
**No washing or pull down
**No washing or pull down
**Should be same as Sample 2 unless RNA is lost during biotin or streptavidin bead steps
**Should be same as Sample 2 unless RNA is lost during biotin or streptavidin bead steps
*Sample 4: RNA template captured by V6 with Ampligase at 37C
*Sample 4: RNA template captured by V6 with Ampligase at 55C
**Negative control to see difference between SplintR and Ampligase
**Negative control to see difference between SplintR and Ampligase
*Sample 5: No template with V6 and SplintR at 37C
*Sample 5: No template with V6 and SplintR at 25C
**NTC checks for ssDNA ligase activity
**NTC checks for ssDNA ligase activity
*Sample 6: biotinylated and strept bead linked RNA captured by V4 padlock probes at 55C followed by wash and then SplintR & gDNA at 37C
*Sample 6: biotinylated and strept bead linked RNA captured by V4 padlock probes at 55C followed by wash and then SplintR & gDNA at 25C
**V4 padlock probes should be targeting wrong strand and completely washed away before
**V4 padlock probes should be targeting wrong strand and completely washed away before
*Sample 7: biotinylated and strept bead linked RNA captured by V6 at 55C followed by wash and then SplintR at 37C
*Sample 7: biotinylated and strept bead linked RNA captured by V6 at 55C followed by wash and then SplintR at 25C


*Summary:
*Summary:
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#Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH
#Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH
#Air-dry for ~5min
#Air-dry for ~5min
#Resuspend pellet in 20ul nf-H2O (or 2X B&W buffer for binding to Streptavidin beads)
#Resuspend pellet in 200ul nf-H2O


===Attach RNA to Streptavidin Beads===
===Attach RNA to Streptavidin Beads===
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**2X B&W Buffer (10 mM Tris-HCl 1 mM EDTA 2M NaCl): 10ml 5M NaCl + 50ul 0.5M EDTA + 62.5ul 4M Tris-HCl + 14.8875ml H2O
**2X B&W Buffer (10 mM Tris-HCl 1 mM EDTA 2M NaCl): 10ml 5M NaCl + 50ul 0.5M EDTA + 62.5ul 4M Tris-HCl + 14.8875ml H2O


#Resuspend beads by 30sec
#Resuspend beads by vortexing 30sec
#Transfer 10ul of beads (10ug/ul) to new tube
#Transfer 100ul of beads (10ug/ul) to new tube
#Add 1ml 1X B&W Buffer and mix
#Add 1ml 1X B&W Buffer and mix
#Pull down by magnet 2min and remove supernatant
#Pull down by magnet 2min and remove supernatant
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#Wash 2x with 100ul Solution A for 2 min each  
#Wash 2x with 100ul Solution A for 2 min each  
#Wash 2x with 100ul Solution B
#Wash 2x with 100ul Solution B
#Resuspend the beads in Solution B
#Resuspend the beads in 200ul 2X B&W Buffer
#Add biotinylated RNA
#Add 200ul biotinylated RNA
#Incubate 15min at RT gently rotating
#Incubate 15min at RT gently rotating
#Pull down with magnet for 3min
#Pull down with magnet for 3min
#Wash 3x with 1X B&W Buffer
#Wash 3x with 1X B&W Buffer
#Resuspend in 1ml 1X PBS
#Resuspend in 60ul nf H2O


===V6 Padlock Probe Capture===
===V6 Padlock Probe Capture===
*SplintR at 37C or 25C? 4hrs or overnight?
*Dilute 2ul of 1ug/ul UHRR into 40ul total(50ng/ul final conc)
*V4 padlock probes: 234nM (10.7ng/ul) from Batch 10
*V6 padlock probes: [[Matt:LabNotes/2015-1-21|854nM]]
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt"  valign="bottom"
| width="65" height="16" | Sample#-RNA
| width="66" | V4 Probes (234nM)
| width="66" | V6 Probes (854nM)
| width="66" | gDNA (80.3ng/ul)
| width="66" | RNA (50ng/ul)
| width="66" | b-RNA (~50ng/ul)
| width="66" | 10X Ampligase Buffer
| width="66" | 10X SplintR Buffer
| width="66" | Ampligase
| width="66" | SplintR
| width="66" | H2O
| width="66" | Total
| width="66" | Overnight Temp
 
|- style="font-size:12pt"  valign="bottom"
| align="right" height="16" | 1
| align="right" | 0
| align="right" | 1
| align="right" | 3.8
| align="right" | 0
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 22.2
| align="right" | 30
| align="right" | 55
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 2-30
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 0.6
| align="right" | 0
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 2
| align="right" | 23.4
| align="right" | 30
| align="right" | 25
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 2-150
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 2
| align="right" | 21
| align="right" | 30
| align="right" | 25
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 2-820
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 16.4
| align="right" | 0
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 2
| align="right" | 7.6
| align="right" | 30
| align="right" | 25
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 3-30
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 0
| align="right" | 0.6
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 2
| align="right" | 23.4
| align="right" | 30
| align="right" | 25
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 3-150
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 2
| align="right" | 21
| align="right" | 30
| align="right" | 25
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 3-820
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 0
| align="right" | 16.4
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 2
| align="right" | 7.6
| align="right" | 30
| align="right" | 25
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 4-30
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 0.6
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0.5
| align="right" | 0
| align="right" | 24.9
| align="right" | 30
| align="right" | 55
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 4-150
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0.5
| align="right" | 0
| align="right" | 22.5
| align="right" | 30
| align="right" | 55
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 4-820
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 16.4
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0.5
| align="right" | 0
| align="right" | 9.1
| align="right" | 30
| align="right" | 55
 
|- style="font-size:12pt"  valign="bottom"
| align="right" height="16" | 5
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 2
| align="right" | 24
| align="right" | 30
| align="right" | 25
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 6-30
| align="right" | 2.5
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 0.6
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 23.9
| align="right" | 30
| align="right" | 55
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 6-150
| align="right" | 2.5
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 21.5
| align="right" | 30
| align="right" | 55
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 6-820
| align="right" | 2.5
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 16.4
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 8.1
| align="right" | 30
| align="right" | 55
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 7-30
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 0
| align="right" | 0.6
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 25.4
| align="right" | 30
| align="right" | 55
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 7-150
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 23
| align="right" | 30
| align="right" | 55
 
|- style="font-size:12pt"  valign="bottom"
| height="16" | 7-820
| align="right" | 0
| align="right" | 1
| align="right" | 0
| align="right" | 0
| align="right" | 16.4
| align="right" | 0
| align="right" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 9.6
| align="right" | 30
| align="right" | 55
 
|}
 
*Added 50ul mineral oil on top
*Magnetic streptavidin beads stick to sides of PCR tube at and above the oil line
*Overnight 21 hrs
 
*Sample 1: Add 3ul Ampligase mix and incubate at 55C for 21 hrs
**1ul Ampligase + 1ul Buffer + 8ul H2O
*Samples 6 and 7:
*#Transfer aqueous phase to new PCR tubes
*#*Beads stuck to original tube so lost a lot during transfer
*#Pull down beads (should have annealed padlock probes attached)
*#Remove supernatant and wash beads twice with 100ul 1X SplintR Buffer
*#Resuspend in 30ul 1X SplintR Buffer with SplintR Ligase (and 300ng gDNA for Samples 6)
*#*Samples 6: 2ul SplintR + 3.8ul gDNA + 3ul 10X Buffer + 21.2ul H2O
*#*Samples 7: 2ul SplintR + 3ul 10X Buffer + 25ul H2O
*#Incubate at 25C for 21 hrs
*#94C for 2min and then hold at 4C for 3 hrs (went to lunch)
*Other Samples: 94C for 2min and then hold at 4C in fridge for 21 hrs
*Take out 10ul from each tube and go straight to qPCR
*Enzyme digest template from ~15ul that is left in tubes
**2ul Exo I/III (1:1) for DNA (Samples 1 & 6)
***37C for 1 hr
**2ul RNaseH and Riboshredder (1:1) for RNA (Samples 2-5 & 7)
***37C for 1 hr
***First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder)
*94C for 2min


===qPCR===
===qPCR===
====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'''
|-
| 1||1||ISB_CA_AF||ISB_CA_AR.T1
|-
| 2||2||ISB_CA_AF||ISB_CA_AR.T2
|-
| 3||3||ISB_CA_AF||ISB_CA_AR.T3
|-
| 4||1||ISB_CA_AF||ISB_CA_AR.T1
|-
| 5||2||ISB_CA_AF||ISB_CA_AR.T2
|-
| 6||3||ISB_CA_AF||ISB_CA_AR.T3
|}-->
====PCR Test for Non-Enzyme Digested Samples====
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
| align="center" style="background:#f0f0f0;"|'''18X Volume'''
|-
| Captured template||1||0
|-
| 10uM ISB_CA_AF||0.4||7.2
|-
| 10uM ISB_CA_AR.T1||0.4||7.2
|-
| 2X KAPA SYBG MM||12.5||225
|-
| H2O||10.7||192.6
|-
| Total||25||432
|}
*Aliquot 24ul from 18X master mix and add 1ul captured template
  Program
  98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
[[Media:SplintR_Ligase_Test_qPCR_-_No_Enzyme_Digest.xlsx|Excel sheet with full PCR data]]<br>
[[File:SplintR_Ligase_Test_qPCR_-_No_Enzyme_Digest.JPG|450px]]
====PCR Test for Enzyme Digested Samples====
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
| align="center" style="background:#f0f0f0;"|'''18X Volume'''
|-
| Captured template||1||0
|-
| 10uM ISB_CA_AF||0.4||7.2
|-
| 10uM ISB_CA_AR.T1||0.4||7.2
|-
| 2X KAPA SYBG MM||12.5||225
|-
| H2O||10.7||192.6
|-
| Total||25||432
|}
*Aliquot 24ul from 18X master mix and add 1ul captured template
  Program
  98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
[[Media:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.xlsx|Excel sheet with full PCR data]]<br>
[[File:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.JPG|450px]]
==Results & Conclusion==
*Pos Ctrl (V6 probes capture gDNA with Ampligase) has low amount of ligated padlock probes
**High Ct in PCR curve
**I guess 300ng gDNA has fewer targets than 30ng RNA
**But same low level as Neg Ctrl (Ampligase used on DNA/RNA hybrid)
*NTC (V6 captures '''nothing''' with SplintR) has same amount of ligated padlock probes as Experimental samples (V6 captures RNA with SplintR)
**suggests SplintR doesn't need RNA splint to ligate DNA
*CONCLUSION: Need to check literature and/or call NEB but it seems SplintR has high ssDNA ligase activity, making it unusable for high specificity padlock probe applications
**Checked and there is no reported ssDNA ligase activity, therefore it must be probes annealing to other probes
**"Template-independent ligation"
===After meeting with Dr. Zhang===
*Reason PosCtrl does not show higher ligation is because forgot 95C is needed to denature gDNA!
*He pointed out there is separation between NTC/30ng and 150ng/820ng PCR curves
**Therefore there is some probe/splint ligation going on and conditions (eg temperature/time/concentration) need to be tuned to increase this separation
===Next Experiment===
*Find shortest amount of time necessary for SplintR Ligase at 37C
**Minimize this time so less non-specific annealing of probes
*Keep constant probe:target ratio for all
*Try 3 NTCs with padlock probe concentrations matching 3 samples
*Annealing temp: 95C 30sec -> Cool to 55C
*Try different annealing times?

Latest revision as of 00:35, 3 September 2016

SplintR Ligase Test[edit]

Reference[edit]

Experimental Outline[edit]

  1. Remove DNA from RNA (UHRR 740000-41)
    • Shouldn't be completely necessary
  2. Biotinylate RNA
  3. Attach RNA to magnetic streptavidin beads
  4. V6 Padlock Probe capture to RNA
  5. Quantify captured products by qPCR
    • Also adds Illumina sequencing adapters

Sample Groups[edit]

  • Use Universal Human Reference RNA (UHRR 740000-41)
  • 3 RNA template concentrations for each (30ng, 150ng and 820ng)
    • For DNA template positive control only do 1 sample using 300ng gDNA
  • Sample 1: gDNA template captured by V6 padlock probes with Ampligase at 55C
    • Standard padlock probe capture except keep at 55C whole time
  • Sample 2: RNA template captured by V6 padlock probes with SplintR at 25C
    • Should be ligated but non-specific because low temp
  • Sample 3: biotinylated and streptavidin bead linked RNA captured by V6 padlock probes with SplintR at 25C
    • No washing or pull down
    • Should be same as Sample 2 unless RNA is lost during biotin or streptavidin bead steps
  • Sample 4: RNA template captured by V6 with Ampligase at 55C
    • Negative control to see difference between SplintR and Ampligase
  • Sample 5: No template with V6 and SplintR at 25C
    • NTC checks for ssDNA ligase activity
  • Sample 6: biotinylated and strept bead linked RNA captured by V4 padlock probes at 55C followed by wash and then SplintR & gDNA at 25C
    • V4 padlock probes should be targeting wrong strand and completely washed away before
  • Sample 7: biotinylated and strept bead linked RNA captured by V6 at 55C followed by wash and then SplintR at 25C
  • Summary:
    • 3 samples with biotinylated RNA
    • 2 samples where RNA is pulled down by magnetic beads and washed to remove non-specific binding

Experiment[edit]

Remove DNA from RNA[edit]

  • Skipped this step

Biotinylate RNA[edit]

  • Use Pierce RNA 3' End Biotinylation Kit (>75% ligation efficiency)
  • 3ul of 1ug/ul UHRR
  1. Thaw all kit components except 30% PEG and DMSO on ice
    • Thaw DMSO at RT and 30% PEG at 37C for 5-10min
  2. Adjust heating block to 85C
  3. Transfer 3ul RNA and 1ul DMSO to 1.5ml tube and heat for 5min at 85C
  4. Place RNA immediately on ice
  5. Prepare labeling reaction mix by adding in order listed
    • RNA volume was < 4ul due to evaporation
    • Total volume < 30ul and probably had some losses when moving to 0.2ml tube because needed thermalcycler for 16C
    • Use new pipette tip to mix reaction mix after PEG addition
Component Volume
nf H2O 4
10X RNA Ligase Buffer 3
Rnase Inhibitor 1
RNA (w/ 25% DMSO) 4
Biotinylated Cytidine (Bis)phosphate 1
T4 RNA Ligase 2
30% PEG 15
Total 30
  1. Incubate at 16C for overnight (~15 hrs)
  2. Add 70ul of nf-H2O
  3. Add 100ul chloroform:isoamyl alcohol to extract RNA ligase
    • 96ul chloroform + 4ul isoamyl alcohol
    • Vortex briefly and then centrifuge 2-3 min at high speed to separate phases
  4. Carefully remove the top (aqueous) phase and transfer to a new tube
  5. Add 10ul 5M NaCl, 1ul glycogen, and 300ul ice-cold 100% EtOH
  6. Precipitate for 1hr at -20C
  7. Centrifuge for 15min at > 13,000 x g at 4C
  8. Remove supernatant
  9. Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH
  10. Air-dry for ~5min
  11. Resuspend pellet in 200ul nf-H2O

Attach RNA to Streptavidin Beads[edit]

  • MyOne C1 protocol
  • Make Solutions A & B and Binding&Wash Buffer
    • Solution A (DEPC-treated 0.1 M NaOH, DEPC-treated 0.05 M NaCl): 2.5ml 1M NaOH + 0.25ml 5M NaCl + 22.25ml nf-H2O
      • NaOH: 39.997g/mol -> 1M NaOH = 0.1g NaOH dissolved in 2.5mL nf H2O
    • Solution B (DEPC-treated 0.1 M NaCl): 0.5ml 5M NaCl + 24.5ml nf-H2O
    • 2X B&W Buffer (10 mM Tris-HCl 1 mM EDTA 2M NaCl): 10ml 5M NaCl + 50ul 0.5M EDTA + 62.5ul 4M Tris-HCl + 14.8875ml H2O
  1. Resuspend beads by vortexing 30sec
  2. Transfer 100ul of beads (10ug/ul) to new tube
  3. Add 1ml 1X B&W Buffer and mix
  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. Wash 2x with 100ul Solution A for 2 min each
  7. Wash 2x with 100ul Solution B
  8. Resuspend the beads in 200ul 2X B&W Buffer
  9. Add 200ul biotinylated RNA
  10. Incubate 15min at RT gently rotating
  11. Pull down with magnet for 3min
  12. Wash 3x with 1X B&W Buffer
  13. Resuspend in 60ul nf H2O

V6 Padlock Probe Capture[edit]

  • Dilute 2ul of 1ug/ul UHRR into 40ul total(50ng/ul final conc)
  • V4 padlock probes: 234nM (10.7ng/ul) from Batch 10
  • V6 padlock probes: 854nM
Sample#-RNA V4 Probes (234nM) V6 Probes (854nM) gDNA (80.3ng/ul) RNA (50ng/ul) b-RNA (~50ng/ul) 10X Ampligase Buffer 10X SplintR Buffer Ampligase SplintR H2O Total Overnight Temp
1 0 1 3.8 0 0 3 0 0 0 22.2 30 55
2-30 0 1 0 0.6 0 0 3 0 2 23.4 30 25
2-150 0 1 0 3 0 0 3 0 2 21 30 25
2-820 0 1 0 16.4 0 0 3 0 2 7.6 30 25
3-30 0 1 0 0 0.6 0 3 0 2 23.4 30 25
3-150 0 1 0 0 3 0 3 0 2 21 30 25
3-820 0 1 0 0 16.4 0 3 0 2 7.6 30 25
4-30 0 1 0 0.6 0 3 0 0.5 0 24.9 30 55
4-150 0 1 0 3 0 3 0 0.5 0 22.5 30 55
4-820 0 1 0 16.4 0 3 0 0.5 0 9.1 30 55
5 0 1 0 0 0 0 3 0 2 24 30 25
6-30 2.5 0 0 0 0.6 0 3 0 0 23.9 30 55
6-150 2.5 0 0 0 3 0 3 0 0 21.5 30 55
6-820 2.5 0 0 0 16.4 0 3 0 0 8.1 30 55
7-30 0 1 0 0 0.6 0 3 0 0 25.4 30 55
7-150 0 1 0 0 3 0 3 0 0 23 30 55
7-820 0 1 0 0 16.4 0 3 0 0 9.6 30 55
  • Added 50ul mineral oil on top
  • Magnetic streptavidin beads stick to sides of PCR tube at and above the oil line
  • Overnight 21 hrs
  • Sample 1: Add 3ul Ampligase mix and incubate at 55C for 21 hrs
    • 1ul Ampligase + 1ul Buffer + 8ul H2O
  • Samples 6 and 7:
    1. Transfer aqueous phase to new PCR tubes
      • Beads stuck to original tube so lost a lot during transfer
    2. Pull down beads (should have annealed padlock probes attached)
    3. Remove supernatant and wash beads twice with 100ul 1X SplintR Buffer
    4. Resuspend in 30ul 1X SplintR Buffer with SplintR Ligase (and 300ng gDNA for Samples 6)
      • Samples 6: 2ul SplintR + 3.8ul gDNA + 3ul 10X Buffer + 21.2ul H2O
      • Samples 7: 2ul SplintR + 3ul 10X Buffer + 25ul H2O
    5. Incubate at 25C for 21 hrs
    6. 94C for 2min and then hold at 4C for 3 hrs (went to lunch)
  • Other Samples: 94C for 2min and then hold at 4C in fridge for 21 hrs
  • Take out 10ul from each tube and go straight to qPCR
  • Enzyme digest template from ~15ul that is left in tubes
    • 2ul Exo I/III (1:1) for DNA (Samples 1 & 6)
      • 37C for 1 hr
    • 2ul RNaseH and Riboshredder (1:1) for RNA (Samples 2-5 & 7)
      • 37C for 1 hr
      • First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder)
  • 94C for 2min

qPCR[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

PCR Test for Non-Enzyme Digested Samples[edit]

Components 1X Volume 18X Volume
Captured template 1 0
10uM ISB_CA_AF 0.4 7.2
10uM ISB_CA_AR.T1 0.4 7.2
2X KAPA SYBG MM 12.5 225
H2O 10.7 192.6
Total 25 432
  • Aliquot 24ul from 18X master mix and add 1ul captured template
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

Excel sheet with full PCR data
File:SplintR Ligase Test qPCR - No Enzyme Digest.JPG

PCR Test for Enzyme Digested Samples[edit]

Components 1X Volume 18X Volume
Captured template 1 0
10uM ISB_CA_AF 0.4 7.2
10uM ISB_CA_AR.T1 0.4 7.2
2X KAPA SYBG MM 12.5 225
H2O 10.7 192.6
Total 25 432
  • Aliquot 24ul from 18X master mix and add 1ul captured template
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

Excel sheet with full PCR data
File:SplintR Ligase Test qPCR - Enzyme Digest.JPG

Results & Conclusion[edit]

  • Pos Ctrl (V6 probes capture gDNA with Ampligase) has low amount of ligated padlock probes
    • High Ct in PCR curve
    • I guess 300ng gDNA has fewer targets than 30ng RNA
    • But same low level as Neg Ctrl (Ampligase used on DNA/RNA hybrid)
  • NTC (V6 captures nothing with SplintR) has same amount of ligated padlock probes as Experimental samples (V6 captures RNA with SplintR)
    • suggests SplintR doesn't need RNA splint to ligate DNA
  • CONCLUSION: Need to check literature and/or call NEB but it seems SplintR has high ssDNA ligase activity, making it unusable for high specificity padlock probe applications
    • Checked and there is no reported ssDNA ligase activity, therefore it must be probes annealing to other probes
    • "Template-independent ligation"

After meeting with Dr. Zhang[edit]

  • Reason PosCtrl does not show higher ligation is because forgot 95C is needed to denature gDNA!
  • He pointed out there is separation between NTC/30ng and 150ng/820ng PCR curves
    • Therefore there is some probe/splint ligation going on and conditions (eg temperature/time/concentration) need to be tuned to increase this separation

Next Experiment[edit]

  • Find shortest amount of time necessary for SplintR Ligase at 37C
    • Minimize this time so less non-specific annealing of probes
  • Keep constant probe:target ratio for all
  • Try 3 NTCs with padlock probe concentrations matching 3 samples
  • Annealing temp: 95C 30sec -> Cool to 55C
  • Try different annealing times?