I usually lose one or two lanes as a result of the bubbling. Is this typical?
Please see photos.
I have no visible issues of leaking. I use fresh APS. I clean my plates rigorously.
Thanks to anyone willing to shed some light on the issue!
Before you pouring the sticking gel you should add Isopropyl alcohol
Try to fill the stacking gel upto the brim and ensure that when placing the comb it is not going in at an angle. I think the bubbles are forming because of uneven displacement of the gel when placing the comb allowing air to trap. Hope it helps!
Hi Justin,
I think you should increase the volume of your stacking gel you prepare because such happen when the stacking gel volume doesn't occupy the space as a result of vigorous mixing that evolve air bubbles due to SDS that foams.When you place you comb in it,you experience having air bubbles and the lanes at the edge are always victims.(1) Prepare 3-4 ml of stacking gel and fill it to the top(2)When placing you comb,proper care must be taken to avoid air bubbles.Since it is SDS-PAGE,normally when you mix together because of the presence of SDS it foams a lot .Whenever you mix,mix gently 1-2 times to avoid excessive foaming and also avoid shortage of volume retained in the tube. Ensure you don't pour foams into the stacking region before putting your comb.I hope this will help.
All the best!
Place your end of blue tip at the corner of plate (right or left), then load your stacking gel carefully until the top of your plate and carefully put your comb (make sure the comb is clean). If still bubbles, just add some mL of IPA.
Hi Justin,
Have you been able to resolve the problem?? I am interested to know the solution for the same problem.
Thanks
I also have this problem! I am very grateful to the one who can solve this problem:)
Hi Jonas Andersson ,
Last year, I also faced the same problem.
Based on my experience, I would like to share the ways to resolve this.
1. Casting Time: If your resolving and/or stacking gel takes more than 15 mins to cast completely, then your APS solution is the problem.
Solution:
- Use freshly prepared APS solution. (Best because APS might be degraded)
- If not, add double the volume of TEMED.
2. After pouring stacking gel, Let it set for one or two mins then place the comb. When you see the bubbles coming from the corner, Immediately place the gel in the running module then pour fresh 1X running or tank buffer inside and used / fresh 1X running buffer outside. After 20 mins you can remove the comb and use.
Thank you KRISHNA KUMAR HARIDHASAPAVALAN !
I will try this next time!
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