NATIVE PAGE - (Oct/28/2005 )

HELLO EVERYONE!

i tried 2 cast native page gel but the mix of components didnt polymerise,it remained in the liquid form.i brought a new APS and TEMED ,and i tried out the native page but still it didnt get polymerised.all de components r prepared freshly .
this is de composition of my stacking and resolving gel

5% stacking gel 10% resolving gel
(make upto 5ml) (make upto 10ml)
water -3.4ml water -4ml
30%acrylamide -1ml 30%acrylamide -3.30ml
1.5Mtris HCl[pH 6.8] -0.7ml 1.5Mtris HCl[pH8.8] -2.50ml
10%APS -100ul 10%APS -100ul
TEMED -5ul TEMED -10ul

i really dont know wat is de problem
pls do help .

thanks in advance.

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I don't know if this is a typo, but if not, would explain the problem:

30%acrylamide -1ml 30%acrylamide -3.30ml
The second component on this line should be bis-acrylamide to get crosslinking.

You'll produce linear polyacrylamide otherwise, which is a viscous liquid rather than a gel.

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HELLO EVERYONE!

i tried 2 cast native page gel but the mix of components didnt polymerise,it remained in the liquid form.i brought a new APS and TEMED ,and i tried out the native page but still it didnt get polymerised.all de components r prepared freshly .
this is de composition of my stacking and resolving gel

i really dont know wat is de problem
pls do help .

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hi phage434

i add bis acrylamide while preparing the 30% acrylamide

the 30% acrylamide(monomer),which i prepare contains :

acrylamide - 4mg/30ml
bis acrylamide - 240mg/30ml

now where have i gone wrong or is there any problem with my composition or reagents.
pls help

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I use 30ul of 10% APS and 30ul of TEMED for 10% resolving gel, then put some isopropanol to flatten the top of the gel and assist the polymerisation. The gel will set around 30min.
Then raise off the isopropanol with water.
For stacking gel, I use 20ul of 10% APS and 20ul of TEMED. The stacking gel will set around 30-40min.

I have tried 30ul of 10%APS and 10ul of TEMED for resolving gel, but it took a whole day to set. (I have no patience to wait that long!!!)

I hope this may help you.

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hi Minnie Mouse

i tried out by increasing the concentration of TEMED upto 40 - 50 ul
and APS upto 200ul .i do add isopropanol to flatten the top .but still it didnt get polymerised.
i have done native page for atleast 5 times but no polymerisation at all.
what should i do?

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maybe you should check your reagents, like TEMEd because I use a very similar composition in my native gel as you do except I use only 5 ul TEMEd and I don't have a problem with polymerisation. Maybe there is a problem with it (TEMED) .Also, maybe while making the 30% acrylamide, the bis-acrylamide got left out. Hope things get settled.

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I think you may have problem with your 30% acrylamide. You may wish to buy the pre-made 30%acrylamide rather than making it yourself.

After I have made the 30%-acrylamide, I put it in a clean bottle and wrap it with foil and put it in 4 degree. I think because it is light-sensitive.

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thanks alot for the advise,i shall try out with both the reagents(TEMED and 30% acrylamide) and see where i have gone wrong.
once again thanks aloooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo
oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooot
Minnie Mouse and soraya

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Hi,
Maybe you should add sodium sulfite to
your gel: Enhances polymerization at
acid pH. Final concentration should
be 3mM
Cheers

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