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S9.6 Slot blotting

KC Karlene A Cimprich
MC Madzia P Crossley

Last updated date: Aug 27, 2020 Views: 1620 Forks: 0

original An abbreviated version of this protocol was published in eLife in Aug, 2016
Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage
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Slot Blot

 

  1. Total nucleic acid was extracted by a standard SDS/Proteinase K lysis followed by phenol/chloroform extraction and EtOH/sodium acetate precipitation. 
  2. Cut Whatman filter paper of appropriate size. Use x3 filter paper per blot.
  3. Cut Hybond N+/Positive nylon membrane (Amersham) to exact size.
  4. Wet membrane for 15 mins in dH2O. 
  5. Put Whatman paper x3 first on slot blot apparatus, then membrane.
  6. Tighten apparatus.
  7. Load 200 uL of dH2O into wells needed.
  8. Switch on vacuum suction and wait for water to go through.
  9. Load 250 ng and 500 ng genomic DNA into wells. Experimental samples should be loaded in duplicate and one well for ssDNA internal control.
  10. For an RNase H-treated control, treat 5ug of DNA with RNase H (NEB) at 37 degrees overnight.
  11. Leave samples on membrane to bind for 30 mins without suction.
  12. After 30 mins, apply gentle suction. Wait until all samples go through.
  13. Open apparatus; cut membrane into two parts, one for RNA-DNA hybrid detection, one for ssDNA detection (for DNA loading control).

For RNA-DNA hybrids

  1. Place membrane DNA side up on tissue paper and cross-link with UV with 1200 J. Select 1200J (0.12J/m2).

For ssDNA (internal controls)

  1. DNA denaturation – cut filter paper to size of membrane. Wet paper with denaturation buffer (0.5 N NaOH; 1.5 M NaCl) until liquid is floating on top. Avoid air bubbles. Place membrane DNA face up on top of filter paper for 10 min at RT. No air bubbles.
  2. DNA neutralization – repeat using neutralization buffer (1M NaCl; 0.5 M Tris-HCL pH7.0), 10 mins at RT.
  3. Place membrane DNA side up on tissue paper and cross-link with UV using auto-crosslinking program with 1200 J (0.12J/m2).
  4. Block both membranes with 5% milk/TBST (0.1% Tween) for 1 hr. 
  5. Incubate the membrane with S9.6 antibody (Kerafast) (1:500 dilution in 1% BSA/TBS Tween 0.1%) overnight at 4 deg.
  6. Incubate membrane for ssDNA with ssDNA mouse antibody 1:10,000 (EMD Millipore MAB 3034, Clone 16–19) in 1% BSA/TBS Tween 0.1%.
  7. Wash membranes 3x TBS 0.1% Tween, 10 min each.
  8. Incubate membranes in 1:10,000 goat anti-mouse HRP in 5% milk/TBS/0.1% Tween at RT for 1 h.
  9. Wash membrane 3x TBS/0.1% Tween, 10 min each.
  10. Detect signal by chemiluminescence.

 

 

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Cimprich, K and Crossley, M P(2020). S9.6 Slot blotting. Bio-protocol Preprint. bio-protocol.org/prep469.
  2. Stork, C. T., Bocek, M., Crossley, M. P., Sollier, J., Sanz, L. A., Chédin, F., Swigut, T. and Cimprich, K. A.(2016). Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage. eLife. DOI: 10.7554/eLife.17548

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