12 replies to this topic

[tigger2010]

Hi, My running gels and stacking gels are not hardening sometimes (or it is taking a long time). Here is my recipe for running gel,
3.3 ml H2O
4 ml Acrylamide
   2.5 ml PH8.8 Tris
  100 ul 10% SDS
  100 ul 10% Aps
   4 ul Temed

Sometimes it works but most of the times it does not polymerize. Why does this happen? If Aps or acrylamide has gone off then it should not polymerize at all times.. somebody please help.  Thanks.

[mdfenko]

the aps or temed could be off (it may still polymerize but would take a long time).

how long do you store the aps solution? we prefer using fresh prepared  but wouldn't use it if stored longer than 1 week at 4C (do not freeze).

how do you mix the solution? if too vigorously then you may be  dissolving oxygen in the solution. oxygen is a free radical scavenger and may slow the polymerization reaction.

[tigger2010]

Thanks very much for your reply.     The Aps we use is an old stock and I have no idea how long it has been there... I make up the solution in a 50 ml tube. It is possible I mixed them too vigorousely. I am doing 2 westerns tomorrow. Will take your advice into account and fingers crossed.

[tigger2010]

Hi
I have made new Aps and tried other people's temed and was extremely careful when making the gel but it still did not polymerize. I have made 6 attemps already! It polymerized in the tube twice but never polymerized between the plates after 30 minutes. What's causing this! Help please. This is so frustrating...

[phage434]

How old is the bottle of persulfate?  Old bottles stop working after a while, and making a new solution with old persulfate will not help.

[mdfenko]

tigger2010, on 27 October 2010 - 02:19 AM, said:

Hi
I have made new Aps and tried other people's temed and was extremely careful when making the gel but it still did not polymerize. I have made 6 attemps already! It polymerized in the tube twice but never polymerized between the plates after 30 minutes. What's causing this! Help please. This is so frustrating...

your plates may not be clean enough.

keep in mind that 30 minutes is usually not enough time for complete polymerization. give it at least an hour.

[Dr_Watson]

When I prepare my APS I make 1mL aliquots that I store in -20C and only keep 1 at 4C. (never had problem with the fact that I stored it at -20C)

You could try to increase the TEMED's volume. With 100uL of APS usualy I use 20uL of Temed.

[hobglobin]

tigger2010, on 26 October 2010 - 07:15 AM, said:

Hi, My running gels and stacking gels are not hardening sometimes (or it is taking a long time). Here is my recipe for running gel,
3.3 ml H2O
4 ml Acrylamide
   2.5 ml PH8.8 Tris
  100 ul 10% SDS
  100 ul 10% Aps
   4 ul Temed

Sometimes it works but most of the times it does not polymerize. Why does this happen? If Aps or acrylamide has gone off then it should not polymerize at all times.. somebody please help.  Thanks.

you wrote that you used acrylamide...hopefully a ready-made mixture containing also bisacrylamide...otherwise you won't have crosslinking and the gel won't polymerise in the way you want

[GNANA]

i normally use 1 to 5 ratio of TEMED/APS...may be bcoz u r using less temed???...

Gnana...

[tigger2010]

Thanks everyone for all your advice. Eventually I used other people's Aps and added 10 ul Temed and it worked! My two gels polymerized very quickly!   Within 30 minutes! Considering that increasing the volume of Temed with my old Aps and my new Aps did not work, I am guessing that my bottle of ammonium perulfate must be too old. Will order a new bottle and make alipuots next time. Still puzzled by the fact that it polymerized in the tube but not in plates.. I cleaned my plates with 70% ethanol.Are dirty plates going to affect polymerization? I am using water or sometimes 70% ethanol as overlay on top of the running gel. I know isopropanol is the proper stuff but water is cheaper. This shouldnt affect anything right? Maybe if I left it for longer at the start it would eventually set? mdfenko, I used to only leave my gel for 30 minutes and it normally worked fine.. does it require 1 hour? If it is taking that long, doesnt it mean the Aps has gone bad?

[mdfenko]

you can add aps and temed in amounts that would allow your gel to polymerize in 5 minutes (or less) but the heat generated would cause convection and the structure would be questionable. also, adding more temed causes shorter strands and a brittle gel (thanks for the info, tfitzwater). we use enough aps and temed so that our gels are nearly fully polymerized within an hour (we let them sit longer to ensure complete polymerization), but we usually pour gradients and need the working time.

what color is your temed? if it is (very) yellow then it may have decomposed too much to continue using the same amount. ideally, you should obtain fresh temed.

aps is somewhat hygroscopic. we store it at room temperature with a silica gel packet in the bottle. for us, it lasts for years.

we overlay with isobutanol because it isn't miscible.

[tigger2010]

Thanks mdfenko thats very informative.Our Temed is a little yellow so we are ordering new Aps, acrylamide and Temed. How do you normally mix your solution by the way? Do you give it a gentle shake?
Thanks.

[mdfenko]

depending on the vessel we mix by swirling or inversion or both (not too vigorously).