Matt:LabNotes/2016-8-21
Jump to navigation
Jump to search
SplintR Ligase Test
Reference
- 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
- Remove DNA from RNA (UHRR)
- Shouldn't be completely necessary
- Biotinylate RNA
- Attach RNA to magnetic streptavidin beads
- V6 Padlock Probe capture to RNA
Remove DNA from RNA
- Use Zymo DNA-Free RNA Kit
- Sample: 4ul of UHRR (1.3 ug/ul)
DNase I Digest
Components | Volume |
UHRR (1.3ug/ul) | 4 |
10X DNase I Buffer | 5 |
DNase I | 2 |
H2O | 39 |
Total | 50 |
- Mix and incubate at 37C for 30min
Purify
- Add 100ul RNA Binding Buffer and mix
- Add 150ul 100% EtOH and mix
- Transfer to Zymo-Spin IC Column in Collection Tube
- Centrifuge at 13,000rcf for 1min and discard flow through
- Add 400ul RNA Prep Buffer
- Centrifuge at 13,000rcf for 1min and discard flow through
- Add 800ul RNA Wash Buffer
- Centrifuge at 13,000rcf for 30sec and discard flow through
- Add 400ul RNA Wash Buffer
- Centrifuge at 13,000rcf for 30sec and discard flow through
- Centrifuge at 13,000rcf for 2min and discard flow through
- Transfer column to 1.5ul tube and add 8ul H2O and let stand for 1min
- Centrifuge at 10,000rcf for 30sec
- Quantify with Nanodrop or Qubit
Biotinylate RNA
- Use Pierce RNA 3' End Biotinylation Kit (>75% ligation efficiency)
- How to verify biotinylated? http://www.ncbi.nlm.nih.gov/pubmed/7814273 maybe
- 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 1ul of 1ug/ul UHRR (RNA) to 1.5ml tube and heat for 3-5min at 85C
- Place RNA immediately on ice
- Prepare labeling reaction mix by adding in order listed
- Use new pipette tip to mix reaction mix after PEG addition
Component | Volume |
nf H2O | 5 |
10X RNA Ligase Buffer | 3 |
Rnase Inhibitor | 1 |
RNA | 3 |
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 20ul nf-H2O (or 2X B&W buffer for binding to Streptavidin beads)
Attach RNA to Streptavidin Beads
- 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
- 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
- Wash 2x with 100ul Solution A for 2 min each
- Wash 2x with 100ul Solution B
- Resuspend the beads in Solution B
- Add biotinylated RNA
- Incubate 15min at RT gently rotating
- Pull down with magnet for 3min
- Wash 3x with 1X B&W Buffer
- Resuspend in 1ml 1X PBS