While pouring both the resolving and the stacking gels inside the glass plates, bubbles are produced because
of the sds due to which the wells do not form properly and so I can't load my samples into them.
Also, does the water layer mixes with the resolving gel (while it solidifies inside the plates) when it is put on the top of it??
Hi Fakhria,
For the bottom layer, I pour water or 0.1% SDS solution on top of it - they do not mix with the gel solution but collect the bubbles well. After the gel is polymerised, you can easily remove it.
If you are experiencing issues with the upper layer, just pour enough of the gel solution so that it would overflow a little and carry the bubbles out.
I hope that helps. Best of luck!
Dear Fakhria Wahid
I agree with Rachel Gad
for the points before pouring the gel solution into glass cassettes. Mixing gel solution using vortex is very important for the better resolution of the blot. While vortex, bubbles may be formed. During the pouring of gel solution you should avoid bubble to come in pipette.Another important things, water is soluble with resolving gel solution so you should not use water to avoid bubbles. To avoid the air bubble 1 ML 100% Isopropanol is best to add after adding running gel solution. After 15-20 min depending upon room temperature you should check remaining gel solution in conical tube for solidification of gel. If gel is solidified then you can remove isopropanol by gently tilting the glass set-up. Then, gently fix the gel comb above the solidified running gel inserting half of its well length. Vortex the stocking gel solution and add it using 1 ML pipette from the gap of each well in the gel comb.
While adding stocking gel avoid adding air bubble. Add the gel solution up to upper edge of the gel glass and gently press the gel comb completely into the stocking gel. If you see any bubbles between the well you can remove gel comb once and again fix it gently.
I hope this will work.
All the best.
Make sure your gel plates are clean and dry. While pipetting the resolving gel into the glass plates, ensure that you aren't taking up bubbles. Once you have poured the resolving gel, cover it gently with a layer of isopropanol. Around twenty minutes later, once the resolving gel has solidifed, tilt the set-up to pour out the isopropanol. Fill up the glass plates up to the brim with your stacking gel and then gently place your comb. A small amount will overflow, taking out bubbles if any.
Hope this helps.
Hey Fakhria,
Do you remove the combs under the running buffer? It looks like you have air bubbles because the running buffer is not drowning the gel completely.
I remove the comb first, then load my samples and after I put the whole thing into the tank, I add the running buffer. And I do drown the gels on the buffer then.
Fakhria Wahid Do it the other way around. First put the cassette in the tank and then fill it up with running buffer. Next remove the comb gently; in case you see newly formed bubbles, flush them out using gel running buffer and a pipette. In the end, start loading your samples.
Rachel, yea these form while I remove the comb. Before I have added the running buffer.
1. Make sure u have added enough volume of resolving gel. Also while inserting your comb, bubbles should be avoided. if bubbles are formed reinsert the comb.
2.once the gel got solidified remove the comb, Have u ever rinsed your gel with water or buffer after the comb was removed???? If not so, try it once. This bubbles may be removed.
3. then fix the plate to apparatus and then fill the tank with running buffer.
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