Yu:Agarose Formaldehyde: Difference between revisions

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==Electrophoresis==
===RNA Sample preparation===


#clean gel box with NaOH and/or SDS, 2 hours to overnight, rinse with water
#Add RNA samples (10µg in nuclease-free water/buffer) to eppi tubes on ice.  The more concentrated the RNA is, the better
#prepare Agarose gel solution [1 % Agarose, 1 x MOPS, H2O to 95 % of end volume]
#Add 1x volume of Ambion Glyoxal sample loading dye (Cat#8551)or [[Yu:Northern_solns#MOPs_Loading_Buffer|MOPs Loading Buffer]] or [[Yu:Northern_solns#Formamide_Loading_Buffer|Formamide Loading Buffer]]. 
#microwave until completely dissolved
##Treat the RNA markers (Invitrogen, cat#15620-016, 0.24-9.5kb, use 3µl per lane) the same way).
#cool down to 60-70 °C, add Formaldehyde (37 %) to 0.6 M end concentration, pour immediately
#Incubate samples for 30 mins at 50°C (make sure you use cap-lock for the eppis to avoid any liquid getting into your samples during incubation in the waterbath)
#allow gel to harden at least 30 min
#Cool on ice for 5-10 mins.
#prepare running buffer [1 x MOPS, 0.2 M Formaldehyde]
#Vortex, pulse-centrifuge to bring the samples down.
#Run the gel - 70 volts for 6 hrs. Dye migrates 1 cm/hr at 50 volts


==Sample preparation==
===Transfer===


#use 5-10 µg total RNA per lane (up to 30 µg)
#Photograph the gel under UV – with a ruler
#bring RNA samples to equal volume (5-10 µl)with DNase/RNase free H<sub>2</sub>O, add same vol. loading buffer
#Trim the gel down to the essential size and wash 2x in copious amounts of water (in a large plastic tray).  Soak in 10X [[Yu:Northern_solns#20x_SSC|SSC]] buffer for 15-30 mins.
#add 0.5 µl EtBr (0.5 µg/µl)
#Cut long blotter paper to the right length, soak in buffer and fold over a glass plate for a buffer bridge in a glass tray.  Place gel on top of it and roll out the bubbles.  Place trimmed films around the gel to ensure that vertical capillary transfer only occurs via the gel area.
#heat for 5 min @ 90 °C, cool on ice
#Cut Hybond N+ membrane to the exact size/slightly smaller.  Wet the membrane in 10X [[Yu:Northern_solns#20x_SSC|SSC]], cover the top of the gel with the membrane and roll out the bubbles.
#Overlay with 2 layers of wet Whatman paper (one layer at a time), roll out the bubbles each time.
#Overlay with 2-2.5” of paper towels (cut to size) or Whatman paper (cut to size).  Cover with a glass plate and 1/2 filled 500 ml bottle and level it.
#Transfer with ample 10X [[Yu:Northern_solns#20x_SSC|SSC]] in the glass tray overnight.
#Mark the wells with pencil before removing and number the lanes.
#Rinse in 2X SSC, dry on filter paper.
#UV-Crosslink in the Stratalinker – autocrosslink, 1-2x.
#Store blot in SaranWrap or go directly to pre-hyb.


==Gel run==
===Hybridization and Wash===


#run gel (8 x 10 cm) in fume hood at 70-100 V (-> 50-70 mA)
#Prehyb 15-30 mins at 65°C
#run until BPB is near the gel end (2.5-3.5 h)
##Pre-warm the buffer
##Pre-hyb using the [[Yu:Northern_solns#Hyb_Buffer|Hyb Buffer]]
#Boil the probe if dsDNA fragments – on the heat block for 2 mins.
  '''Use CAPS LOCK and caution!'''
  '''Make sure you monitor the area for any radioactive contamination.'''
#Discard the pre-hyb buffer and add 10 ml of new hyb buffer, add the probe into the hyb tube and hyb O/N at 65°C rotating'''
====Wash====
#Discard probe (or store in 50ml conical)
#Add 50 ml of 2X SSC/0.1% SDS for a quick rinse.  Discard.
#Add approx. 50 ml of 2X SSC/0.1% SDS, 15 mins at 65°C rotating.
#Add approx. 50 ml of 1X SSC/0.1% SDS, 15 mins at 65°C rotating.
#Add approx. 50 ml of 0.5X SSC/0.1% SDS, 15 mins at 65°C rotating
#Wrap in seran wrap (moist but not wet).  Expose on the phosphorimager screen


==Northern transfer of RNA==
===Stripping the Membrane===
 
Boil 500 ml of water (with 2 ml of 20% SDS), add into a tray, put the membrane and gentle rock for 30 mins.  Discard water (down the sink is ok), and repeat (2-3x) until small/none of the signal can be detected by Geiger counter.
#soak gel 3 times 5 min in distilled water (to remove Formaldehyde)
#photogragh gel with ruler beside it
#cut GeneScreen membrane (Nylon, DuPont) to exact gel size
#soak membrane in water for a few seconds
#set up capillary blot with 10 x SSC transfer buffer:
##2 wet Whatman - gel - membrane - 2 wet Whatman - 2 dry Whatman – paper towel - glassplate - weight
#transfer 16-24 h with changes of the paper towel
#mark lanes, remove membrane, wash briefly in 2 x SSC
#place membrane on wet Whatman paper and UV-crosslink damp (auto crosslink setting, 254 nm, Stratagene, Stratalinker)
#bake membrane @ 80 °C for 1-2 h
 
==Hybridization==
 
#prehybridize membrane for 1-4 h @ 42 °C with 5-10 ml prehybridization buffer
#heat radioactive labeled probe for 3 min @ 95 °C, cool on ice
#discard prehybridization buffer, add hybridization buffer and probe, incubate ON @ 42 °C
#wash membrane 1 x 15 min with 2 x SSC @ RT
#wash with 2 x SSC, 0.1 % SDS @ 65 °C until background is low
#ash with 0.1 x SSC, 0.1 x SDS @ 65 °C (optional)
#expose wet membrane under saran wrap (-80 °C)
#important: never let the membrane dry (until the blot is stripped)
 
==Stripping and re-hybridization==
 
#wash membrane for 30 min to 3 h in strip solution @ 75 - 85 °C until no radioactivity can be detected on the membrane
#membrane can now be air dried and stored @ RT
#for re-hybridization (up to 10 times) follow the hybridization protocol

Latest revision as of 08:42, 11 September 2009

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RNA Sample preparation

  1. Add RNA samples (10µg in nuclease-free water/buffer) to eppi tubes on ice. The more concentrated the RNA is, the better
  2. Add 1x volume of Ambion Glyoxal sample loading dye (Cat#8551)or MOPs Loading Buffer or Formamide Loading Buffer.
    1. Treat the RNA markers (Invitrogen, cat#15620-016, 0.24-9.5kb, use 3µl per lane) the same way).
  3. Incubate samples for 30 mins at 50°C (make sure you use cap-lock for the eppis to avoid any liquid getting into your samples during incubation in the waterbath)
  4. Cool on ice for 5-10 mins.
  5. Vortex, pulse-centrifuge to bring the samples down.
  6. Run the gel - 70 volts for 6 hrs. Dye migrates 1 cm/hr at 50 volts

Transfer

  1. Photograph the gel under UV – with a ruler
  2. Trim the gel down to the essential size and wash 2x in copious amounts of water (in a large plastic tray). Soak in 10X SSC buffer for 15-30 mins.
  3. Cut long blotter paper to the right length, soak in buffer and fold over a glass plate for a buffer bridge in a glass tray. Place gel on top of it and roll out the bubbles. Place trimmed films around the gel to ensure that vertical capillary transfer only occurs via the gel area.
  4. Cut Hybond N+ membrane to the exact size/slightly smaller. Wet the membrane in 10X SSC, cover the top of the gel with the membrane and roll out the bubbles.
  5. Overlay with 2 layers of wet Whatman paper (one layer at a time), roll out the bubbles each time.
  6. Overlay with 2-2.5” of paper towels (cut to size) or Whatman paper (cut to size). Cover with a glass plate and 1/2 filled 500 ml bottle and level it.
  7. Transfer with ample 10X SSC in the glass tray overnight.
  8. Mark the wells with pencil before removing and number the lanes.
  9. Rinse in 2X SSC, dry on filter paper.
  10. UV-Crosslink in the Stratalinker – autocrosslink, 1-2x.
  11. Store blot in SaranWrap or go directly to pre-hyb.

Hybridization and Wash

  1. Prehyb 15-30 mins at 65°C
    1. Pre-warm the buffer
    2. Pre-hyb using the Hyb Buffer
  2. Boil the probe if dsDNA fragments – on the heat block for 2 mins.
 Use CAPS LOCK and caution!
 Make sure you monitor the area for any radioactive contamination.
  1. Discard the pre-hyb buffer and add 10 ml of new hyb buffer, add the probe into the hyb tube and hyb O/N at 65°C rotating

Wash

  1. Discard probe (or store in 50ml conical)
  2. Add 50 ml of 2X SSC/0.1% SDS for a quick rinse. Discard.
  3. Add approx. 50 ml of 2X SSC/0.1% SDS, 15 mins at 65°C rotating.
  4. Add approx. 50 ml of 1X SSC/0.1% SDS, 15 mins at 65°C rotating.
  5. Add approx. 50 ml of 0.5X SSC/0.1% SDS, 15 mins at 65°C rotating
  6. Wrap in seran wrap (moist but not wet). Expose on the phosphorimager screen

Stripping the Membrane

Boil 500 ml of water (with 2 ml of 20% SDS), add into a tray, put the membrane and gentle rock for 30 mins. Discard water (down the sink is ok), and repeat (2-3x) until small/none of the signal can be detected by Geiger counter.

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