The body part (or bottom part) of stacking gel was polymerized very well. However, when I took out the comb gently, some of the wells were not forming well, causing the volume that I load the sample decreased.
Any techniques or tips when pouring the stacking gel?
Besides, I'm still new to this platform.. Can anyone tell me how to tag the person who replied me?
Thanks Killol,
I did prepare the excess stacking gel, and I saw it is solidified in the tube. However some of the wells were collapsed even I removed the comb slowly
Prepare APS fresh and double its concentration (20%) for stacking gel.
good advice all around here, especially the fresh TEMED bit. A million years ago when I was a biochemist, I used 'home-made' apparatus with gel spacers that were likely a bit thicker (1mm) than the commercial stuff folks use these days. I used an HPLC syringe to put the samples into the wells, but if necessary, I would use the fine needle on the end of the syringe to reposition the 'legs' first, to make sure they were now properly and vertically aligned before adding the samples to the wells.
Thanks for the answer provided..
Hi Dr.Sutherland.. What did you say is to align the well properly before you loading the sample?
Hi Killol.. Beaker is too large for preparing the stacking gel I wonder.. Usually I prepare the stacking gel in 15mL tube and there will be always 3~4mL leave in the tube.. Although I can see that the gel is already polymerized well (little bit of water on top of it), some of the wells are not forming well
your issues seem to be that the stacking gel is not polymerizing in a timely manner. Others have identified the most likely causes, so check that the acrylamide concentration is correct, and that you are using fresh APS and that the TEMED is not past its 'use-by' date. Try allowing the stacking gel and few extra minutes to see if this resolves the problem. My advice was more concerned with ensuring that the 'legs' of stacking gel that separate the wells from each other are properly aligned, and you can use a fine needle to straighten them, should they get bent over when you extract the 'comb'. If after extracting the comb, there is still some liquid stacking gel in the bottom of the well, you can use a syringe with a fine needle to flush out the stacking gel buffer before it polymerizes and solidifies in the bottom of the well.
Hi.. The same thing happened to me when I left the gel for too long to set. Usually after adding the stacking gel, leave it to polymerise for at least 15 minutes, then take out the comb slowly and rinse the wells with tap water. and put the comb back.
Also I noticed that the same thing happened when I put too much resolving gel, so I didn't have enough room for the comb. So the comb just barely touched the (already solidified) resolving gel. And the well formed was really messed up. So just be careful to leave enough space for the stacking gel.
I hope this helps!
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