Matt:LabNotes/2016-8-21: Difference between revisions
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Program | Program | ||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min | 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==== | ====PCR Test for Enzyme Digested Samples==== | ||
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Program | Program | ||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min | 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: | [[File:SplintR_Ligase_Test_qPCR_-_Enzyme_Digest.JPG|450px]] | ||
====PCR | ==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]
- V4 and V7 Capture Experiment
- V4 Capture Analysis
- Purify UHRR of DNA
- V6 & V8 Probe Production
- Linking biotin to streptavidin
- NEED to add extra wash steps to remove RNases!
Experimental Outline[edit]
- Remove DNA from RNA (UHRR 740000-41)
- Shouldn't be completely necessary
- Biotinylate RNA
- Attach RNA to magnetic streptavidin beads
- V6 Padlock Probe capture to RNA
- 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
- Thaw all kit components except 30% PEG and DMSO on ice
- Thaw DMSO at RT and 30% PEG at 37C for 5-10min
- Adjust heating block to 85C
- Transfer 3ul RNA and 1ul DMSO to 1.5ml tube and heat for 5min at 85C
- Place RNA immediately on ice
- 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 |
- Incubate at 16C for overnight (~15 hrs)
- Add 70ul of nf-H2O
- 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
- Carefully remove the top (aqueous) phase and transfer to a new tube
- Add 10ul 5M NaCl, 1ul glycogen, and 300ul ice-cold 100% EtOH
- Precipitate for 1hr at -20C
- Centrifuge for 15min at > 13,000 x g at 4C
- Remove supernatant
- Wash pellet with 300ul ice-cold 70% EtOH and remove EtOH
- Air-dry for ~5min
- 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
- 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
- Resuspend beads by vortexing 30sec
- Transfer 100ul of beads (10ug/ul) to new tube
- Add 1ml 1X B&W Buffer and mix
- Pull down by magnet 2min and remove supernatant
- Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant
- Wash 2x with 100ul Solution A for 2 min each
- Wash 2x with 100ul Solution B
- Resuspend the beads in 200ul 2X B&W Buffer
- Add 200ul biotinylated RNA
- Incubate 15min at RT gently rotating
- Pull down with magnet for 3min
- Wash 3x with 1X B&W Buffer
- 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:
- 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)
- Transfer aqueous phase to new PCR tubes
- 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)
- 2ul Exo I/III (1:1) for DNA (Samples 1 & 6)
- 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?