Yu:Agarose Formaldehyde: Difference between revisions

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#cool down to 60-70 °C, add Formaldehyde (37 %) to 0.6 M end concentration, pour immediately
#cool down to 60-70 °C, add Formaldehyde (37 %) to 0.6 M end concentration, pour immediately
#allow gel to harden at least 30 min
#allow gel to harden at least 30 min
#prepare running buffer [1 x MOPS, 0.2 M Formaldehyde]
#prepare running buffer: 1x [[Yu:Northern_solns#10x_MOPs|MOPs]], 0.2 M Formaldehyde


==Sample preparation==
==Sample preparation==


#use 5-10 µg total RNA per lane (up to 30 µg)
#use 5-10 µg total RNA per lane (up to 30 µg)
#bring RNA samples to equal volume (5-10 µl)with DNase/RNase free H<sub>2</sub>O, add same vol. loading buffer
#bring RNA samples to equal volume (5-10 µl) with DNase/RNase free H<sub>2</sub>O, add an equal volume of [[Yu:Northern_solns#Loadng_Buffer|loading buffer]]
#add 0.5 µl EtBr (0.5 µg/µl)
#add 0.5 µl EtBr (0.5 µg/µl)
#heat for 5 min @ 90 °C, cool on ice
#heat for 5 min @ 90 °C, cool on ice
Line 28: Line 28:
#cut GeneScreen membrane (Nylon, DuPont) to exact gel size
#cut GeneScreen membrane (Nylon, DuPont) to exact gel size
#soak membrane in water for a few seconds
#soak membrane in water for a few seconds
#set up capillary blot with 10 x SSC transfer buffer:
#set up capillary blot with 10x [[Yu:Northern_solns#20x_SSC|SSC]] transfer buffer
##2 wet Whatman - gel - membrane - 2 wet Whatman - 2 dry Whatman – paper towel - glassplate - weight
##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
  '''Important: Roll out any air bubbles before applying dry Whatman
#mark lanes, remove membrane, wash briefly in 2 x SSC
#transfer 16-24 h, changing the paper towel if necessary
#place membrane on wet Whatman paper and UV-crosslink damp (auto crosslink setting, 254 nm, Stratagene, Stratalinker)
#mark lanes, remove membrane, wash briefly in 2x [[Yu:Northern_solns#20x_SSC|SSC]]
#bake membrane @ 80 °C for 1-2 h
#place membrane on wet Whatman paper and UV-crosslink damp (auto crosslink setting, 254 nm, Stratagene, Stratalinker) or bake membrane @ 80 °C for 1-2 h


==Hybridization==
==Hybridization==


#prehybridize membrane for 1-4 h @ 42 °C with 5-10 ml prehybridization buffer
#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
#Heat radioactive labeled probe for 3 min @ 95 °C, cool on ice
#discard prehybridization buffer, add hybridization buffer and probe, incubate ON @ 42 °C
#Discard prehybridization buffer, add hybridization buffer and probe, incubate ON @ 42 °C
#wash membrane 1 x 15 min with 2 x SSC @ RT
#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
#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)
#Wash with 0.1 x SSC, 0.1 x SDS @ 65 °C (optional)
#expose wet membrane under saran wrap (-80 °C)
#Expose wet membrane under saran wrap (-80 °C)
#important: never let the membrane dry (until the blot is stripped)
  '''Important: never let the membrane dry (until the blot is stripped)'''


==Stripping and re-hybridization==
==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
#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
#membrane can now be air dried and stored @ RT
#for re-hybridization (up to 10 times) follow the hybridization protocol
#for re-hybridization (up to 10 times) follow the hybridization protocol

Revision as of 15:41, 10 September 2009

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Electrophoresis

  1. clean gel box with NaOH and/or SDS, 2 hours to overnight, rinse with water
  2. prepare Agarose gel solution [1 % Agarose, 1 x MOPS, H2O to 95 % of end volume]
  3. microwave until completely dissolved
  4. cool down to 60-70 °C, add Formaldehyde (37 %) to 0.6 M end concentration, pour immediately
  5. allow gel to harden at least 30 min
  6. prepare running buffer: 1x MOPs, 0.2 M Formaldehyde

Sample preparation

  1. use 5-10 µg total RNA per lane (up to 30 µg)
  2. bring RNA samples to equal volume (5-10 µl) with DNase/RNase free H2O, add an equal volume of loading buffer
  3. add 0.5 µl EtBr (0.5 µg/µl)
  4. heat for 5 min @ 90 °C, cool on ice

Gel run

  1. run gel (8 x 10 cm) in fume hood at 70-100 V (-> 50-70 mA)
  2. run until BPB is near the gel end (2.5-3.5 h)

Northern transfer of RNA

  1. soak gel 3 times 5 min in distilled water (to remove Formaldehyde)
  2. photogragh gel with ruler beside it
  3. cut GeneScreen membrane (Nylon, DuPont) to exact gel size
  4. soak membrane in water for a few seconds
  5. set up capillary blot with 10x SSC transfer buffer
    1. 2 wet Whatman - gel - membrane - 2 wet Whatman - 2 dry Whatman – paper towel - glassplate - weight
 Important: Roll out any air bubbles before applying dry Whatman
  1. transfer 16-24 h, changing the paper towel if necessary
  2. mark lanes, remove membrane, wash briefly in 2x SSC
  3. place membrane on wet Whatman paper and UV-crosslink damp (auto crosslink setting, 254 nm, Stratagene, Stratalinker) or bake membrane @ 80 °C for 1-2 h

Hybridization

  1. Prehybridize membrane for 1-4 h @ 42 °C with 5-10 ml prehybridization buffer
  2. Heat radioactive labeled probe for 3 min @ 95 °C, cool on ice
  3. Discard prehybridization buffer, add hybridization buffer and probe, incubate ON @ 42 °C
  4. Wash membrane 1 x 15 min with 2 x SSC @ RT
  5. Wash with 2 x SSC, 0.1 % SDS @ 65 °C until background is low
  6. Wash with 0.1 x SSC, 0.1 x SDS @ 65 °C (optional)
  7. Expose wet membrane under saran wrap (-80 °C)
 Important: never let the membrane dry (until the blot is stripped)

Stripping and re-hybridization

  1. wash membrane for 30 min to 3 h in strip solution @ 75-85 °C until no radioactivity can be detected on the membrane
  2. membrane can now be air dried and stored @ RT
  3. for re-hybridization (up to 10 times) follow the hybridization protocol
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