Polyacrylamide gel not polymerize - (Jul/26/2005 )

hi, I've a stupid question...
I've always used pre-cast polyacrylamide gel for analyze restriction fragment after digestion, but now I'd like to prepare the gel by myself, the problem is that I'm not able to polymerize the polyacrilamide...don't know why. I've used the recipe that a fried give me, as follow:

stock sol of:
-acryl:bisacryl 29:1, 40% 87 ml
-TBE 10X 50 ml
-glicerol 50 ml
-distilled water up to 500 ml

then for 7% poly gel
5 ml of stock sol
20 microliter of APS 10%
11 microliter of TEMED

nothing happen also after couple of hours!!!!
I've tryied to rise the amount of APS and TEMED (250-25 or 35 700) same result no polymerization!!!!

please somebody can help me!!!!

thank you so much

this is my recipe for polyacrylamide gel used for analyze restriction fragment after digestion

-H2O 7.4ml
-TBE 10x 1
-acryl/bis 1.5ml (40%)
-APS 10% 100µl
-TEMED 10µl

NB: i think glycerol makes the gel to polymerize longer but i never use it for this kind of experiment
i have never made any stok solution, i alway prepare the mix before my electrophoresis
and renew your 10% APS stock frequently!

good luck ;-)

hi
usually, fresh APS should be used.
You use APS : 1:100 total vol
TEMED : 1 : 1000 total vol
i've never used glycerol...

Addding glycerol make the acrylamide less brittle without interfering with it separation capabilities.

have a look here:

http://www.protocol-online.org/forums/inde...pic=4893&hl=aps

mike

thank'a lot...the gel is perfect...but just another question...
of course very stupid...the final concentration of my gel with your recipe is 18%?

ciao