Home  Contacts  Order  Catalog  Support  Search  Alphabetical Index  Numerical Index Restriction Enzymes Modifying Enzymes PCR & RT-PCR Molecular Cloning Nucleic Acid Purification Molecular Labeling & Detection In vitro Transcription Electrophoresis Products Nucleotides Transfection Reagents Reagents A N S W E R S

Frequently Asked Questions: RiboRuler™ RNA Ladders (4)

RiboRuler™ RNA ladders and other RNA related products 2X RNA Loading Dye RNA quantification on gels RNA electrophoresis: Is it necessary to heat RiboRuler™ RNA ladders prior to loading on denaturing gels? What gels are most suitable for each RiboRuler™ RNA ladder? Which type of RNA electrophoresis is most appropriate for my application? What is the difference between formaldehyde and glyoxal denaturing agarose gel electrophoresis? How should the quality of the RNA electrophoresis be evaluated? Northern blots and labeling

1. Is it necessary to heat RiboRuler™ RNA ladders prior to loading on denaturing gels?

To ensure complete RNA denaturation, it is always necessary to heat RNA ladders at 70°C for 10 min and to chill on ice prior to loading onto gels.

2. What gels are most suitable for each RiboRuler™ RNA ladder?

RiboRuler™ RNA Ladders, High Range show the best performance on:

• native 0.8-1.5% agarose gels with TAE buffer
• denaturing formaldehyde 0.8-1.5% agarose gels with MOPS buffer
• denaturing glyoxal/DMSO 0.8-1.5% agarose gels with sodium phosphate buffer

RiboRuler™ Low Range RNA Ladders show the best performance on:

• native 1.7-2.5% agarose gel with TAE buffer
• denaturing formaldehyde 1.7-2.5% agarose gels with MOPS buffer
• denaturing glyoxal/DMSO 1.7-2.5% agarose gels with sodium phosphate buffer
• denaturing 4-8% polyacrylamide gels with TBE buffer

3. Which type of RNA electrophoresis is most appropriate for my application?

• Native RNA electrophoresis is typically used to asses the overall quality of total RNA.
  After electrophoresis of total RNA samples in the presence of ethidium bromide, the 28S and 18S human rRNA should be clearly visible under UV illumination. Fast-migrating bands composed of 5.8S RNA and 5S RNA can also be visible, depending on the RNA purification procedure. The intensity of the 28S RNA should be approximately twice the intensity of the 18S RNA. The 28S human rRNA band migrates approximately with the 5000 b band, the 18S human rRNA band migrates approximately with the 1900 b band.
• Denaturing agarose or polyacrylamide gel electrophoresis is traditionally used for more precise sizing of RNA. However, the 2X RNA Loading Dye supplied with RiboRuler™ RNA ladders contains the denaturing agent formamide, which permits precise sizing of RNA molecules even during native gel electrophoresis.
• For Northern blots, to ensure the most complete denaturation of RNA, it is recommended to perform formaldehyde agarose gel electrophoresis in MOPS buffer.

4. What is the difference between formaldehyde and glyoxal denaturing agarose gel electrophoresis?

Both formaldehyde gel electrophoresis in MOPS buffer and the glyoxal/DMSO gel electrophoresis in sodium phosphate buffer are comparable and best used to separate RNA according to their size.

• Formaldehyde forms unstable schiff bases with the imino-groups of the RNA guanine residues. These adducts maintain RNA in the denatured state by preventing intra-molecular Watson-Crick base pairing.
• The two aldehyde groups of glyoxal react under slightly acidic conditions with the imino-groups of the RNA guanine residues to form cyclic derivatives that prevent intrastrand Watson-Crick base pairing.

5. How should the quality of the RNA electrophoresis be evaluated?

• The bands of RiboRuler™ RNA ladders should appear sharp.
• After electrophoresis of total RNA samples in the presence of ethidium bromide, the 28S and 18S human rRNA bands should be clearly visible under UV illumination.
• Fast-migrating bands composed of 5.8S RNA and 5S RNA can also be visible, depending on the RNA purification procedure.
• The intensity of the 28S RNA band should be approximately twice the intensity of the 18S RNA band..
• The band of 28S human rRNA migrates approximately with the 5000 b band, and the 18S human rRNA migrates approximately with the 1900 b band.

Home

Search

Contacts

Order

Catalog

Support

Updated vasario 01, 2008 09:49