what could cause gel not to polymerize (solidify)? - gel making (Jun/23/2008 )
Hi everyone. My running gels are not hardening. I've added
6 mL 30%acrylamide/0.8%bisacrylamide,
3.75 mL pH8.8 3M tris HCl,
100 uL 20% SDS
5 mL water
50 uL APS
10 uL TEMED
Am I missing anything? Do any of the above reagents require storage at a particular temperature prior to use? Why are my gels not solidifying like they used to?
HELP,
Miles
6 mL 30%acrylamide/0.8%bisacrylamide,
3.75 mL pH8.8 3M tris HCl,
100 uL 20% SDS
5 mL water
50 uL APS
10 uL TEMED
Am I missing anything? Do any of the above reagents require storage at a particular temperature prior to use? Why are my gels not solidifying like they used to?
HELP,
Miles
The commonest troublemaker is APS. Make fresh APS and store it in 4'C for a short time, -20'C for a long time. Everytime, gel stops polymerising,make fresh!
I will try it and get back to you...u are potentially my day saver cellcounter!
6 mL 30%acrylamide/0.8%bisacrylamide,
3.75 mL pH8.8 3M tris HCl,
100 uL 20% SDS
5 mL water
50 uL APS
10 uL TEMED
Am I missing anything? Do any of the above reagents require storage at a particular temperature prior to use? Why are my gels not solidifying like they used to?
HELP,
Miles
The commonest troublemaker is APS. Make fresh APS and store it in 4'C for a short time, -20'C for a long time. Everytime, gel stops polymerising,make fresh!
Cellcounter is right. APS is the main culprit. It's very important that your store your dry APS in a dessicator. It will go bad over time if you don't (it happened to me!)
Clare
You guys were right on the money. As soon as I remade APS, I used fresh APS and what doyou know? My gel was rock hard(exaggerating) within 30 minutes.
thanks much,
grateful lab technician!
thanks much,
grateful lab technician!
Awesome
6 mL 30%acrylamide/0.8%bisacrylamide,
3.75 mL pH8.8 3M tris HCl,
100 uL 20% SDS
5 mL water
50 uL APS
10 uL TEMED
Am I missing anything? Do any of the above reagents require storage at a particular temperature prior to use? Why are my gels not solidifying like they used to?
HELP,
Miles
i've read somewhere that mixing the gel mixture around too much can cause oxygen saturation which then impedes gel polymerization...any truth in that?
6 mL 30%acrylamide/0.8%bisacrylamide,
3.75 mL pH8.8 3M tris HCl,
100 uL 20% SDS
5 mL water
50 uL APS
10 uL TEMED
Am I missing anything? Do any of the above reagents require storage at a particular temperature prior to use? Why are my gels not solidifying like they used to?
HELP,
Miles
i've read somewhere that mixing the gel mixture around too much can cause oxygen saturation which then impedes gel polymerization...any truth in that?
That is true. But unless you mix it like a nut, it should eventually polymerise.
Atmospheric oxygen is a free radical scavenger that scavanges SO4- ions produced by the inherently unstable APS necessary for the polymerization.
In the good old days, people also used to deaerate acrylamide mix to make gels polymerize.
I was still deaerating the acrylamide mix to get the oxygen out from the solution last year (just in case). But now I found out it is not a necessity anymore. They can still polymerize even without deaeration!