Difference: DenaturingFormaldehydeGels (3 vs. 4)

Revision 42018-04-29 - SimonDAlton

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META TOPICPARENT name="ProtocolList"

Denaturing Formaldehyde Gels for Verifying RNA size

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 RUNNING GEL
  1. Load samples as usual for a gel.
  2. The buffer for this run is 1x MOPS, diluted in water (does not have to be DEPC-H2O).
Changed:
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  1. Run at a reduced voltage of 70V in order to avoid heating the gel.
>
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  1. Run at a reduced voltage of 80V in order to avoid heating the gel.
  STAINING GEL
Changed:
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  1. Remove the gel from the rig and wash quickly once in 1x TAE, followed by a longer, 20 minute wash in 1x TAE. This will bring the pH to a more neutral value to allow Sybrgold to bind to RNA.
>
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  1. Remove the gel from the rig when the blue loading dye is approximately 1/2-3/4 down the gel. If the predicted RNA size is 4kb or larger, leave gel til 3/4 to get good resolution, smaller RNAs can be left for the shorter time.
  2. Wash quickly once in 1x TAE, followed by a longer, 20 minute wash in 1x TAE. This will bring the pH to a more neutral value to allow Sybrgold to bind to RNA.
 
  1. Incubate gel in 1x Sybrgold (from 10k x stock) in 1x TAE for 30 mins.
  2. Image as usual.
 
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