Difference: DenaturingFormaldehydeGels (5 vs. 6)

Revision 62018-11-09 - SimonDAlton

Line: 1 to 1
 
META TOPICPARENT name="ProtocolList"

Denaturing Formaldehyde Gels for Verifying RNA size

Line: 23 to 23
  GEL
  1. Dissolve 0.5g of agarose in 43.5ml of DEPC-treated water as you would for usual agarose gel.
Changed:
<
<
  1. Allow this solution to cool as you would normally do before pouring. You can run it under the tap to speed this up. Note that you will add cold 10x MOPS at the next step, and that this solution seems to set more quickly than in 1x TAE.
>
>
  1. Allow this solution to cool as you would normally do before pouring. You can run it under the tap to speed this up.
 
  1. IN HOOD add 1.5ml of 37% formaldehyde and 5 ml of 10x MOPS.
  2. Pour in to cast with comb, leave to set.
Line: 32 to 32
 
  1. Add water to reach Xul total volume
  2. Add 3 volumes of NorthernMax buffer.
  3. The Millennium Markers need to be denatured also. Use 2ul + 6ul NorthernMax buffer.
Changed:
<
<
  1. Heat at 65C for 15 minutes. IMPORTANT When this has finished, immediately move samples to ice. If samples are allowed to slowly cool, some RNAs may bind to proteins/form secondary structure and will get stuck in the wells of the gel.
>
>
  1. Heat at 65C for 15 minutes. IMPORTANT When this has finished, immediately move samples to ice. If samples are allowed to slowly cool, some RNAs may bind to proteins/form secondary structure and will run weird.
 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).
 
This site is powered by the TWiki collaboration platform Powered by Perl This site is powered by the TWiki collaboration platformCopyright ©2022 Barrick Lab contributing authors. Ideas, requests, problems? Send feedback