RNA formaldehyde gels: Difference between revisions
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Clean gel box and combs etc with detergent, then dionized water.
Then incubate the gel box and components in 10% H2O2 (hydrogen peroxide, stored in cold room) for 30 minutes.
Rinse box thoroughly with DEPC water.
Your should pour (and ideally run) the gel in the hood, since the gel contains formaldehyde. [see protocol from Maniatis (original edition)—attached.]
Heat your samples (see step 3 Maniatis protocol) for 15 minutes at 65 or 70 C and then put immediately on ice. The add loading buffer (see step 4 Maniatis).
ALSO, pre-run the gel at 50 V for 10 minutes
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<li>Your should pour (and ideally run) the gel in the hood, since the gel contains formaldehyde. [see protocol from Maniatis (original edition)—attached.] | <li>Your should pour (and ideally run) the gel in the hood, since the gel contains formaldehyde. [see protocol from Maniatis (original edition)—attached.] | ||
<h3>Running the gel:</h3> | <h3>Running the gel:</h3> | ||
Just before loading | <i>Just before loading the samples:</i> | ||
<li>Heat your samples (see step 3 Maniatis protocol) for 15 minutes at 65 or 70 C and then put immediately on ice. The add loading buffer (see step 4 Maniatis). | <li>Heat your samples (see step 3 Maniatis protocol) for 15 minutes at 65 or 70 C and then put immediately on ice. The add loading buffer (see step 4 Maniatis). | ||
<li>ALSO, pre-run the gel at 50 V for 10 minutes | <li>ALSO, pre-run the gel at 50 V for 10 minutes | ||
<ul> | |||
<li>Load samples. | <li>Load samples. | ||
<li>For mini gels, run at 30V for 4-5 hours. (for best results place the gel on a bed of ice to keep it cold) | <li>For mini gels, run at 30V for 4-5 hours. (for best results place the gel on a bed of ice to keep it cold) | ||
<li>Stain in EtBr 1 hour to overnight ( 5 µl of 10 mg/ml solution per 50 ml DEPC water) | <li>Stain in EtBr 1 hour to overnight ( 5 µl of 10 mg/ml solution per 50 ml DEPC water) | ||
<li>Destain in DEPC water for 1-2 hours (change water 2x during this time) | <li>Destain in DEPC water for 1-2 hours (change water 2x during this time) | ||
<li>Take picture (Karla's lab has a good digital documentation system) | <li>Take picture (Karla's lab has a good digital documentation system) | ||
</ul> | |||
Revision as of 12:21, 10 March 2014
Prepare gel box:
For making gel and preparing samples:
Running the gel:
Just before loading the samples:
- Load samples.
- For mini gels, run at 30V for 4-5 hours. (for best results place the gel on a bed of ice to keep it cold)
- Stain in EtBr 1 hour to overnight ( 5 µl of 10 mg/ml solution per 50 ml DEPC water)
- Destain in DEPC water for 1-2 hours (change water 2x during this time)
- Take picture (Karla's lab has a good digital documentation system)