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

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*Use Pierce RNA 3' End Biotinylation Kit (>75% ligation efficiency)
*Use Pierce RNA 3' End Biotinylation Kit (>75% ligation efficiency)
*How to verify biotinylated? http://www.ncbi.nlm.nih.gov/pubmed/7814273 maybe
*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
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:11pt"  valign="bottom"
| width="51" height="14" | Component
| width="51" | Volume
|- style="font-size:11pt"  valign="bottom"
| height="14" | nf H2O
| align="right" | 5
|- style="font-size:11pt"  valign="bottom"
| height="14" | 10X RNA Ligase Buffer
| align="right" | 3
|- style="font-size:11pt"  valign="bottom"
| height="14" | Rnase Inhibitor
| align="right" | 1
|- style="font-size:11pt"  valign="bottom"
| height="14" | RNA
| align="right" | 3
|- style="font-size:11pt"  valign="bottom"
| height="14" | Biotinylated Cytidine (Bis)phosphate
| align="right" | 1
|- style="font-size:11pt"  valign="bottom"
| height="14" | T4 RNA Ligase
| align="right" | 2
|- style="font-size:11pt"  valign="bottom"
| height="14" | 30% PEG
| align="right" | 15
|- style="font-size:11pt"  valign="bottom"
| height="14" | Total
| align="right" | 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===
===Attach RNA to Streptavidin Beads===

Revision as of 02:28, 22 August 2016

SplintR Ligase Test

Reference

Experimental Outline

  1. Remove DNA from RNA (UHRR)
    • Shouldn't be completely necessary
  2. Biotinylate RNA
  3. Attach RNA to magnetic streptavidin beads
  4. 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

  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 1ul of 1ug/ul UHRR (RNA) to 1.5ml tube and heat for 3-5min at 85C
  4. Place RNA immediately on ice
  5. 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
  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 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
  1. Resuspend beads by vortexing
  2. Transfer 100ul of beads (10ug/ul) to new tube
  3. Pull down by magnet 2min and remove supernatant
  4. Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant
  5. Wash 2x with 100ul Solution A for 2 min each
  6. Wash 2x with 100ul Solution B
  7. Resuspend the beads in Solution B
  8. Add biotinylated RNA
  9. Incubate 15min at RT gently rotating
  10. Pull down with magnet for 3min
  11. Wash 3x with 1X B&W Buffer
  12. Resuspend in 1ml 1X PBS

V6 Padlock Probe Capture