RNA formaldehyde gels: Difference between revisions

From Tanaka Wiki
Jump to navigation Jump to search
No edit summary
No edit summary
 
(3 intermediate revisions by the same user not shown)
Line 1: Line 1:
<h3>Prepare gel box:</h3>
== Procedure ==
<li>Clean gel box and combs etc with detergent, then dionized water.
=== Prepare gel box: ===
<li>Then incubate the gel box and components in 10% H2O2 (hydrogen peroxide, stored in cold room)  for 30 minutes.   
* Clean gel box and combs etc with detergent, then dionized water.
<li>Rinse box thoroughly with DEPC water.
* Then incubate the gel box and components in 10% H2O2 (hydrogen peroxide, stored in cold room)  for 30 minutes.   
<h3>For making gel and preparing samples:</h3>
* Rinse box thoroughly with DEPC water.
<li>Your should pour (and ideally run) the gel in the hood,  since the gel contains formaldehyde. [see protocol from Maniatis (original edition)—attached.]
=== For making gel and preparing samples: ===
<h3>Running  the gel:</h3>
* Your should pour (and ideally run) the gel in the hood,  since the gel contains formaldehyde. [see protocol from Maniatis (original edition)—attached.]
Just before loading the samples:
=== Running  the gel: ===
<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).
''Just before loading the samples:''
<li>ALSO, pre-run the gel at 50 V for 10 minutes
* 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
<li>Load samples.
<ul>
 
* 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)
* 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)
<li>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)
<li>Destain in DEPC water  for 1-2 hours (change water  2x during this time)
</ul>
 
<li>Take picture  (Karla's lab has a good digital documentation system)

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)
Retrieved from "https://wiki.tanakalab.org/index.php?title=RNA_formaldehyde_gels&oldid=727"