I need a PROTOCOL !!!!

Rover Rover at livenet.net
Tue Aug 20 19:36:22 EST 1996


baez at chem.columbia.edu (Victor Anaya-Baez) wrote:

>Does anyone have a protocol for Electrophoresis through an agarose gel
>containing methylmercuric hydroxide or formaldehyde? I also need a
>protocol for Electrophoresis through agarose gel for RNA that hasbee
>denaturalized with glyoxal and dimethylsulfoxide. Your help will be
>greatly appreciated. Thank you!

>baez at chem.columbia.edu

	I gather you are going to electrophorese RNA in the first question.
I use a  formaldehyde/agarose gel, not wanting to deal with mercury
toxicity, and it works fine.  You can find protocols for both your
questions in "Current Protocols in Molecular Biology".

The formaldehyde gel recipe I use is for a 1.2% agarose which is the
standard we use. (DEPC treat anything that will contact your RNA)

Agarose 2.4 g
water 143 mls

melt in microwave or boiling bath.

add 37 mls formaldehyde (37%soln)
add 20 mls 10 MAE or Mops

(optional) add 25 ul of a 5-10 mg/ml Ethidium Bromide 

-allow to cool to hand comfortable and pour. ( We use a 200 ml
 gel in a submersible apparatus, scale based upon your own 
equipment.

The running buffer is 1X MAE or MOPS.

Sample prep.
We denature our samples  according to the following:	

RNA 5ul
Formamide 10 ul (molecular grade)
formaldehyde 3.5 ul
10X MAE 2 ul
10X loading dye (25% glycerol) 2ul

incubate at 65 C for 15 minutes and place on ice until 
ready to load.

You voltage will vary depending on equipment, we generally
go as low as possible and overnight, but you can run quicker
during the day with minimal loss of clarity.

I don't use glyoxal...sorry

hope this helps,






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