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 [ | #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 | #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 | #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 | '''Important: Roll out any air bubbles before applying dry Whatman | ||
#mark lanes, remove membrane, wash briefly in | #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]] | ||
#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 | ||
# | #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 | ||
# | #Wash 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== | ==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
Electrophoresis
- clean gel box with NaOH and/or SDS, 2 hours to overnight, rinse with water
- prepare Agarose gel solution [1 % Agarose, 1 x MOPS, H2O to 95 % of end volume]
- microwave until completely dissolved
- 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
- prepare running buffer: 1x MOPs, 0.2 M Formaldehyde
Sample preparation
- 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 H2O, add an equal volume of loading buffer
- add 0.5 µl EtBr (0.5 µg/µl)
- heat for 5 min @ 90 °C, cool on ice
Gel run
- run gel (8 x 10 cm) in fume hood at 70-100 V (-> 50-70 mA)
- run until BPB is near the gel end (2.5-3.5 h)
Northern transfer of RNA
- 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 10x SSC transfer buffer
- 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
- transfer 16-24 h, changing the paper towel if necessary
- mark lanes, remove membrane, wash briefly in 2x SSC
- 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
- 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
- Wash 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