I want to cast my gels more efficiently. At the moment, I need to create cocktails according to a recipe and to do this takes time.
In my current casting routine, I mix milliQ, 1.5M TRIS, 30% acrylamide and 20% SDS, finish it off with TEMED and ammonium persulfate and cast.
To work more efficiently, my idea is to make a big batch of milliQ, 1.5M TRIS, 30% acrylamide and 20% SDS, of which I can sample my needed volume whenever I need to cast a gel and then add TEMED.
My question is if this is reliable.
Hi,
If i get your question clearly,preparing large stock and aliquot into small volume for each reagent so as to prepare your recipe for SDS-PAGE is not a bad idea but i will suggest to you that each aliquot should be properly kept at 4 degree and anytime you need it,you need to degas/allow to normalize to room temperature before use.My question is this why using 20% SDS?I think 10% SDS is okay for you.Whenever you need to prepare your recipe of any % of SDS then use them and add Ammonium per sulfate and TEMED. Never add ammonium per sulfate for a long time and later add TEMED because it might form gel in your test tube before use.This method stated above is reliable and efficient.
I hope this will help you.
All the best!
Hi Ijsbrand,
I would not make a mix of MQ, Tris, Acrylamid and SDS and store that.
Making stocksolutions of each of them is ok, as stated by Adesola.
With kind regards,
Ronald
Thank you both for your response!
Adesola
You understood correctly, I desire to make a large stock so that I can aliquote ~10mL for my gels each time I need it, and add TEMED and APS directly to induce polymerization and cast. Up till now I had really nice gel/blot results but I just want to cut the time. Stock SDS comes in 20%, and for 20mL SDS-PAGE I use 0.1 mL 20% SDS
Hi Ijsbrand,
We were experiencing with the same thing in our lab regarding SDS-PAGE gel casting. You can make your mixture of AA, Tris and SDS but I would not store it for too long in the fridge. The resolution of the PAGE than is getting poor. So make it, but use it within 1, at max. 2 weeks. Of course, the mixture has to be stored at 4 degrees.
Vladena
Chinmaya, I do not have any issues with my APS. I prefer to use freshly aliquoted (500 microliters) 10% APS from -20 and store leftovers at 4 if colleagues need any the same day. Personally, I still have good results with APS at 4 degrees.
My question was only to verify if fellow researchers were familiar with making AA, TRIS, SDS, MilliQ mastermixes to save time with casting. Indeed, before you cast (and only then) you need to add TEMED and APS in order to obtain polymerization.
Thank you Vladena for you information. I will experiment in my spare time if the mastermixes still give good results. However, chrystalized SDS won't help :P
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