Difference between revisions of "RNA formaldehyde gels"
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(Created page with "<h3>Prepare gel box: <li>Clean gel box and combs etc with detergent, then dionized water. <li>Then incubate the gel box and components in 10% H2O2 (hydrogen peroxide, stored i...") |
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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)