Difference between revisions of "RNA formaldehyde gels"
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| − | + | == Procedure == | |
| − | + | === Prepare gel box: === | |
| − | + | * 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. | ||
| + | === For making gel and preparing samples: === | ||
| + | * Your should pour (and ideally run) the gel in the hood, since the gel contains formaldehyde. [see protocol from Maniatis (original edition)—attached.] | ||
| + | === Running the gel: === | ||
| + | ''Just before loading the samples:'' | ||
| + | * 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 | ||
| + | <ul> | ||
| + | * 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) | ||
| + | </ul> | ||
Latest revision as of 10:54, 27 March 2015
Procedure
Prepare gel box:
- 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.
For making gel and preparing samples:
- Your should pour (and ideally run) the gel in the hood, since the gel contains formaldehyde. [see protocol from Maniatis (original edition)—attached.]
Running the gel:
Just before loading the samples:
- 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
- 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)