Denaturing acrylamide gel purification of nucleic acids

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (17:10, 11 January 2008) (view source)
(Still adding to the protocol)
 
(3 intermediate revisions not shown.)
Line 1: Line 1:
-
===Denature===
+
==Curators==
-
Add urea to 50% in the gel.
+
''Anyone should feel free to add themselves as a curator for this consensus protocol.  You do not need to be a curator in order to contribute.''
 +
 
 +
==Abstract==
 +
The presence of the denaturant urea in the gel prevents the formation of secondary structure when purifying RNA or DNA.  This protocol can be used to resolve and purify RNA or DNA oligos between 2 and 300 basepairs in length.  Analytical or preparative scale gels may be used, depending on the amount of material available and whether one desires to carry it forward.
 +
 
 +
==Materials==
 +
===Reagents===
 +
#Acrylamide solution (for safety and convenience, SequaGel reagents may be used)
 +
#Urea solution (again, SequaGel is a good source)
 +
#''N,N,N',N'''-Tetramethylethylenediamine (TEMED)
 +
#10% (w/v) ammonium persulfate (APS) in water
 +
##Store TEMED and APS at 4˚C
 +
#10x TBE buffer
 +
##Tris base (108 g)
 +
##Boric acid (55 g)
 +
##0.5 M EDTA at pH 8.0 (40 mL)
 +
##Deionized/distilled water to total vol. 1 L
 +
##Note: 10x TBE may take some time to dissolve, even with fast stirring
 +
#RNA or DNA to be purified
 +
#Solid urea (60.06 g/mol)
 +
 
 +
===Equipment===
 +
#Gel casting equipment
 +
##Glass plates
 +
##Plastic separators
 +
##Plastic comb (to form wells)
 +
##Fastener to hold plates and separators together (binder clips may be used)
 +
#Power supply capable of wattage control
 +
#UV radiation source
 +
#UV reactive silica TLC plates in waterproof material (sheet protector may be used)
 +
#Serological pipettor
 +
#Pipettors
 +
 
 +
==Procedure==
 +
A step by step guide to the experimental procedure.
 +
 
 +
==Critical steps==
 +
*RNA secondary structure can strongly impact how RNA electrophoreses through the gel. Therefore, electrophoresis of RNA is usually done under denaturing conditions.  However, to simply assess the presence of RNA and its quality, a native gel might be sufficient. <cite>LabGuidetoRNA</cite>
 +
*The choice of gel matrix depends on the size range of RNAs to be analyzed.  Use 3-20% polyacrylamide for RNAs < 500bp.  For RNAs between 0.5-8.0 kb, use 1.5% denaturing agarose gel.  For a larger size range (typically necessary for Northern analysis), use 1.0-1.2% denaturing agarose gel.
 +
 
 +
==Troubleshooting==
 +
*RNases are the biggest problem in RNA work.  [[Avoiding RNase contamination|Use proper precautions]].
 +
 
 +
==Notes==
 +
*For best resolution, pour gels as thin as possible (0.5-0.75cm is typical especially for efficient blotting later) and run at low voltage. <cite>RNAmethodologies</cite>
 +
*[[Tom Knight]] strongly recommends using glyoxal denaturation rather than formaldehyde denaturation due to the safety issues of formaldehyde.
 +
*Many protocols call for recirculation of buffer during electrophoresis of glyoxylated RNA.  However, recent electrophoresis buffers (like 10X BPTE electrophoresis buffer) are more stable and do not require this. <cite>MolecularCloning1</cite>
 +
 
 +
==Acknowledgments==
 +
 
 +
 
 +
==References==
 +
<biblio>
 +
#MolecularCloning1 [[doi:10.1101/pdb.prot4057|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]]
 +
#MolecularCloning2 [[doi:10.1101/pdb.prot4050|Molecular Cloning: Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde]]
 +
#RNAmethodologies isbn=0-12-249695-7
 +
#CurrentProtocols [[doi:10.1002/0471142727.mb0409s67|Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment]]
 +
#Ambion [http://www.ambion.com/techlib/append/supp/rna_gel.html Agarose Gel Electrophoresis of RNA] from Ambion
 +
#LabGuidetoRNA isbn=0-471-12536-9
 +
#ProtocolsOnline [http://www.protocol-online.org/prot/Molecular_Biology/RNA/RNA_Electrophoresis/index.html RNA electrophoresis]
 +
</biblio>
 +
 
 +
==Specific Protocols==
 +
#Specific protocol here.
 +
 
 +
[[Category:Protocol]]
 +
[[Category:RNA]]
 +
[[Category:DNA]]

Current revision

Contents

Curators

Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute.

Abstract

The presence of the denaturant urea in the gel prevents the formation of secondary structure when purifying RNA or DNA. This protocol can be used to resolve and purify RNA or DNA oligos between 2 and 300 basepairs in length. Analytical or preparative scale gels may be used, depending on the amount of material available and whether one desires to carry it forward.

Materials

Reagents

  1. Acrylamide solution (for safety and convenience, SequaGel reagents may be used)
  2. Urea solution (again, SequaGel is a good source)
  3. N,N,N',N'-Tetramethylethylenediamine (TEMED)
  4. 10% (w/v) ammonium persulfate (APS) in water
    1. Store TEMED and APS at 4˚C
  5. 10x TBE buffer
    1. Tris base (108 g)
    2. Boric acid (55 g)
    3. 0.5 M EDTA at pH 8.0 (40 mL)
    4. Deionized/distilled water to total vol. 1 L
    5. Note: 10x TBE may take some time to dissolve, even with fast stirring
  6. RNA or DNA to be purified
  7. Solid urea (60.06 g/mol)

Equipment

  1. Gel casting equipment
    1. Glass plates
    2. Plastic separators
    3. Plastic comb (to form wells)
    4. Fastener to hold plates and separators together (binder clips may be used)
  2. Power supply capable of wattage control
  3. UV radiation source
  4. UV reactive silica TLC plates in waterproof material (sheet protector may be used)
  5. Serological pipettor
  6. Pipettors

Procedure

A step by step guide to the experimental procedure.

Critical steps

  • RNA secondary structure can strongly impact how RNA electrophoreses through the gel. Therefore, electrophoresis of RNA is usually done under denaturing conditions. However, to simply assess the presence of RNA and its quality, a native gel might be sufficient. [1]
  • The choice of gel matrix depends on the size range of RNAs to be analyzed. Use 3-20% polyacrylamide for RNAs < 500bp. For RNAs between 0.5-8.0 kb, use 1.5% denaturing agarose gel. For a larger size range (typically necessary for Northern analysis), use 1.0-1.2% denaturing agarose gel.

Troubleshooting

Notes

  • For best resolution, pour gels as thin as possible (0.5-0.75cm is typical especially for efficient blotting later) and run at low voltage. [2]
  • Tom Knight strongly recommends using glyoxal denaturation rather than formaldehyde denaturation due to the safety issues of formaldehyde.
  • Many protocols call for recirculation of buffer during electrophoresis of glyoxylated RNA. However, recent electrophoresis buffers (like 10X BPTE electrophoresis buffer) are more stable and do not require this. [3]

Acknowledgments

References

  1. isbn:0-471-12536-9. [LabGuidetoRNA]
  2. isbn:0-12-249695-7. [RNAmethodologies]
  3. Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels [MolecularCloning1]
  4. Molecular Cloning: Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde [MolecularCloning2]
  5. Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment [CurrentProtocols]
  6. Agarose Gel Electrophoresis of RNA from Ambion [Ambion]
  7. RNA electrophoresis [ProtocolsOnline]

Specific Protocols

  1. Specific protocol here.
Personal tools