78
FENG QIAN AND GREGORY G. GERMINO
Preparing an
agarose/formaldehyde gel
stranded, and this must be eliminated if their molecular weights are to be
accurately determined. Therefore, RNA is usually denatured using one of
the methods described below prior to loading on a gel, and the gel itself is
also run under denaturing conditions.
Procedure
Denaturation of RNA using formaldehyde
RNA is denatured at 65°C in a formamide-formaldehyde buffer for 15 min,
and then loaded onto a 0.8 to 1.2o/o agarose gel containing formaldehyde.
Polyacrylamide gels can be used when smaller molecules are to be resolved.
Denaturation of RNA using glycoxal and DMSO
Sampies are incubated in a glycoxal/DMSO buffer for 1 h at 50°C prior to
loading onto the gel. The gel is prepared with 10 mM NaH2P04, pH 7 running buffer. Recirculation of the buffer during the electrophoresis is required to keep the pH of the bufferat 7. At pH of 8 or greater, the glycoxal
dissociates from the RNA. (Methyl mercuric hydroxide is also an excellent
RNA denaturant, but its toxicity has limited its use.)
Note: An important point discussed in the section on RNA isolation needs
to be re-emphasized in this section: RNases are ubiquitous and stable, and
RNA preparations are thus prone to be degraded. Either an electrophoresis
apparatus should be used that is solely dedicated to RN A electrophoresis, or
it should be treated with 0.5 N NaOH and 10 mM EDTA solution prior to
each use.
RNA protocols
The agarose concentration of a Northern gel is typically 0.8 -1.2o/o. For 100
ml of a 1 o/o gel, weigh out 1 g agarose, add 10 mllOxMOPS buffer and 73 ml
H20. Heat the mix in microwave to dissolve agarose. Pour the hot solution
into a 100 ml graduated cylinder and make the volume to 83 ml, if necessary.
Leave the solution in a fume cupboard until it reaches approximately 60°C,
then add 17 ml of37o/o formaldehyde, swirl to mix (avoid bubbles) and immediately pour into the gel tray.
FENG QIAN AND GREGORY G. GERMINO
Preparing an
agarose/formaldehyde gel
stranded, and this must be eliminated if their molecular weights are to be
accurately determined. Therefore, RNA is usually denatured using one of
the methods described below prior to loading on a gel, and the gel itself is
also run under denaturing conditions.
Procedure
Denaturation of RNA using formaldehyde
RNA is denatured at 65°C in a formamide-formaldehyde buffer for 15 min,
and then loaded onto a 0.8 to 1.2o/o agarose gel containing formaldehyde.
Polyacrylamide gels can be used when smaller molecules are to be resolved.
Denaturation of RNA using glycoxal and DMSO
Sampies are incubated in a glycoxal/DMSO buffer for 1 h at 50°C prior to
loading onto the gel. The gel is prepared with 10 mM NaH2P04, pH 7 running buffer. Recirculation of the buffer during the electrophoresis is required to keep the pH of the bufferat 7. At pH of 8 or greater, the glycoxal
dissociates from the RNA. (Methyl mercuric hydroxide is also an excellent
RNA denaturant, but its toxicity has limited its use.)
Note: An important point discussed in the section on RNA isolation needs
to be re-emphasized in this section: RNases are ubiquitous and stable, and
RNA preparations are thus prone to be degraded. Either an electrophoresis
apparatus should be used that is solely dedicated to RN A electrophoresis, or
it should be treated with 0.5 N NaOH and 10 mM EDTA solution prior to
each use.
RNA protocols
The agarose concentration of a Northern gel is typically 0.8 -1.2o/o. For 100
ml of a 1 o/o gel, weigh out 1 g agarose, add 10 mllOxMOPS buffer and 73 ml
H20. Heat the mix in microwave to dissolve agarose. Pour the hot solution
into a 100 ml graduated cylinder and make the volume to 83 ml, if necessary.
Leave the solution in a fume cupboard until it reaches approximately 60°C,
then add 17 ml of37o/o formaldehyde, swirl to mix (avoid bubbles) and immediately pour into the gel tray.