Fong:RNA gel electrophoresis/Denaturing

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(New page: ==Overview== '''This protocol is under construction''' Gel electrophoresis separates RNA in essentially the same way as DNA, but since RNA often folds into a native conformation, it is ne...)
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# Autoclave bottles on liquid cycle or for 10min at 120°C to remove traces of DEPC.  
# Autoclave bottles on liquid cycle or for 10min at 120°C to remove traces of DEPC.  
# Spray all glassware, casting tray, and gel box with RNAse Away and rinse well with DEPC treated water.
# Spray all glassware, casting tray, and gel box with RNAse Away and rinse well with DEPC treated water.
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===Denature RNA in buffer===
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# Combine:
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#* 5μL RNA sample (up to 30μg)
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#* 1μL 10x MOPS
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#* 3.5μL formaldehyde
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#* 10μL formamide
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# Incubate at 60°C for 15-20min
===Make Denaturing Gel (medium, 50mL gel)===
===Make Denaturing Gel (medium, 50mL gel)===
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# Add 3.5μL ethidium bromide, and pour into casting tray.
# Add 3.5μL ethidium bromide, and pour into casting tray.
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===Denature RNA in buffer===
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===Loading and running gel===
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5μL of the orange formaldehyde loading dye works for 1-30μg RNA.  Modify amounts of RNA sample and dye to get strong enough bands, depending on the sample type and experiment requirements.  Run the gel at 120V for 45-50min, or, for better resolution, 100V for 1hr.
==Notes==
==Notes==
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
-
#List troubleshooting tips here. 
 
-
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
 
-
#Anecdotal observations that might be of use to others can also be posted here. 
 
-
 
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-
 
-
==References==
 
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==Contact==
==Contact==
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*Who has experience with this protocol?
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[[User:Yu Deng| Deng]] </br>
 +
[[User:Christopher M Gowen| Chris]]
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  

Revision as of 13:56, 16 April 2009

Contents

Overview

This protocol is under construction

Gel electrophoresis separates RNA in essentially the same way as DNA, but since RNA often folds into a native conformation, it is necessary to denature the RNA strands if separation by size is needed. The RNA is denatured in this protocol by incubating with fomaldehyde at 60°C.

As with all RNA work, great care must be taken to avoid any contamination with RNAse.

Materials

  • Agarose
  • DEPC
  • 10x MOPS buffer, made using DEPC-treated water (see below)
  • Formaldehyde
  • Formamide
  • RNAse Away spray
  • RNAse free filter pipette tips
  • Gel casting tray and comb
  • Gel box
  • Electrophoresis power supply

Procedure

Treating water with DEPC to remove RNAse

  1. For 1% DEPC solution, add 2ml DEPC to 2L bottle and fill with nanopure H2O.
  2. Shake well and allow solution to incubate for at least 4 hours, or overnight if possible.
  3. Autoclave bottles on liquid cycle or for 10min at 120°C to remove traces of DEPC.
  4. Spray all glassware, casting tray, and gel box with RNAse Away and rinse well with DEPC treated water.

Denature RNA in buffer

  1. Combine:
    • 5μL RNA sample (up to 30μg)
    • 1μL 10x MOPS
    • 3.5μL formaldehyde
    • 10μL formamide
  2. Incubate at 60°C for 15-20min

Make Denaturing Gel (medium, 50mL gel)

  1. In a DEPC-H2O rinsed flask, combine
    • 0.5g Agarose
    • 4.2mL 10x MOPS
    • 37.5mL DEPC-H2O
    • 3.7μL formaldehyde
  2. Microwave for ~1.5 min until melted, cool to ~60°C
  3. Add 3.5μL ethidium bromide, and pour into casting tray.

Loading and running gel

5μL of the orange formaldehyde loading dye works for 1-30μg RNA. Modify amounts of RNA sample and dye to get strong enough bands, depending on the sample type and experiment requirements. Run the gel at 120V for 45-50min, or, for better resolution, 100V for 1hr.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

Contact

Deng </br> Chris

or instead, discuss this protocol.


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