9. RNA is less stable than DNA and should not be repeatedly
freeze–thawed. To ensure long-term stability, we store our
RNA stocks at À80
C aliquoted in TE buffer.
10. Where possible, non-intercalating fluorescent dyes should be
used to label nucleic acids. We use SYBR Safe stain for DNA as
studies have demonstrated that SYBR Safe is less mutagenic
than ethidium bromide.
11. The site of the fluorescent label can be 5
0 , 3
0 or internal. The
choice of labeling site will depend on the characteristics of the
nucleic acid; this should be chosen to minimize interference
with RNA structure or protein binding.
12. If labeling is carried out after chemical synthesis or in vitro
transcription of the nucleic acid, the efficiency of labeling must
be assessed. This can be determined by comparing the molarity
of the fluorescent label (by absorbance of the fluorophore) to
the molarity of the nucleic acid (by absorbance at 260 nm).
This is carried out to ensure that there is sufficient fluorescently
labeled nucleic acid for later detection.
13. Typhoon FLA Imagers (GE) enable the detection of fluorescently stained, fluorescently labeled, or radioactively labeled
(phosphor imaging screen) nucleic acids at high spatial resolution. The compatibility of the Typhoon lasers/emission filters
and the selected fluorophore should be confirmed prior to
beginning the experiment.
14. Once the gel-casting apparatus has been assembled, it can be
checked for leakage. Pour 7 ml of isopropanol between the
glass plates and monitor the meniscus level; if the meniscus falls
over time, there is a leak and the gel caster should be reassembled. Pour away the isopropanol if there are no leakages.
Ensure that the plates are dry before continuing with gel
polymerization.
15. The choice of comb depends on spacer width, number of
samples, and sample volume. We typically use Bio-Rad MiniPROTEAN glass plates with a 1 mm spacer and combs with
either 10 or 15 wells. These correspond to maximum sample
volumes of roughly 26 μl and 44 μl, respectively.
16. Air bubbles introduce undesirable smears in the gel and should
be avoided. In our experience, pouring the gel mixture to the
top of the apparatus so that there is spill over and then inserting
the comb at an angle reduces the chance of bubbles.
17. In our experience, although gels will appear to polymerize
within ~10 min, gels run more uniformly and with sharper
bands if left for at least an hour, presumably since the crosslinked matrix is more homogenous.
EMSAs to Detect Protein-DNA/RNA Interactions
333
freeze–thawed. To ensure long-term stability, we store our
RNA stocks at À80
C aliquoted in TE buffer.
10. Where possible, non-intercalating fluorescent dyes should be
used to label nucleic acids. We use SYBR Safe stain for DNA as
studies have demonstrated that SYBR Safe is less mutagenic
than ethidium bromide.
11. The site of the fluorescent label can be 5
0 , 3
0 or internal. The
choice of labeling site will depend on the characteristics of the
nucleic acid; this should be chosen to minimize interference
with RNA structure or protein binding.
12. If labeling is carried out after chemical synthesis or in vitro
transcription of the nucleic acid, the efficiency of labeling must
be assessed. This can be determined by comparing the molarity
of the fluorescent label (by absorbance of the fluorophore) to
the molarity of the nucleic acid (by absorbance at 260 nm).
This is carried out to ensure that there is sufficient fluorescently
labeled nucleic acid for later detection.
13. Typhoon FLA Imagers (GE) enable the detection of fluorescently stained, fluorescently labeled, or radioactively labeled
(phosphor imaging screen) nucleic acids at high spatial resolution. The compatibility of the Typhoon lasers/emission filters
and the selected fluorophore should be confirmed prior to
beginning the experiment.
14. Once the gel-casting apparatus has been assembled, it can be
checked for leakage. Pour 7 ml of isopropanol between the
glass plates and monitor the meniscus level; if the meniscus falls
over time, there is a leak and the gel caster should be reassembled. Pour away the isopropanol if there are no leakages.
Ensure that the plates are dry before continuing with gel
polymerization.
15. The choice of comb depends on spacer width, number of
samples, and sample volume. We typically use Bio-Rad MiniPROTEAN glass plates with a 1 mm spacer and combs with
either 10 or 15 wells. These correspond to maximum sample
volumes of roughly 26 μl and 44 μl, respectively.
16. Air bubbles introduce undesirable smears in the gel and should
be avoided. In our experience, pouring the gel mixture to the
top of the apparatus so that there is spill over and then inserting
the comb at an angle reduces the chance of bubbles.
17. In our experience, although gels will appear to polymerize
within ~10 min, gels run more uniformly and with sharper
bands if left for at least an hour, presumably since the crosslinked matrix is more homogenous.
EMSAs to Detect Protein-DNA/RNA Interactions
333