Hi everybody... I am starting in molecular markers. I have problems running microsatellites into a poliagrylamide gel. I`ve the Biorad`s sequi-Gen GT electroforesis cell. When I inject gel solution into the glass plate, I cant avoid bubble formation. I hope that someone have been using this electrophotesis cell and can give me some tips to use it.
Hi,
The following injection times (from the bottom of IPC to the top) were found to result
in bubble-free gels: for 50 cm gels with 0.4 mm spacers, between 40–45 seconds; for
50 cm gels with 0.25 spacers, between 50–65 seconds. Injection times of 10 seconds
or less can result in bubble formation in the gel.
• Bubbles can form at the gel front because of soiled areas or uneven siliconization or
coating of the glass plates.
• To achieve bubble free gels, thoroughly clean both plates and siliconize the outer glass
plate before each use.
• If bubbles begin to form at the gel front, hard tapping on top of the IPC assembly
(above the bubble formation) while slowly injecting the gel solution should eliminate
the bubble. Alternatively, the comb end of the IPC assembly can be momentarily
lifted at an angle to facilitate elimination.
or follow the link :-
http://www.cmu.edu.cn/cmu/upl_files/327/2007111485016635.pdf
Hope this will help.
add some more glycerol to the loading buffer. It increase density as well as reduces the bubble formation
hi, u can remove the bubbel formation. I have not used biorad apparatus, but works on PAGE. first clean the plates with soap very nicely. then clean it with ethanol with tissue very properly, see in light from the edge, if any dirt or tissue paper is left, clean more, till u see no pasrticles.
Cleaning is main to avoid bubbles.
Then load your gel immediately after setting the apparatus.
I think biorad one is horizontal one. so seal it with brown tape and clamps from all side. u can also remove bubble while loading the gel by tilting it upward slightly as bubbles come up and then disappears (dont tilt the glass plate more as gel will leak then) .
Hoefer company is having vertical electrophoresis for page where u have to load the gel vertically so no problem of bubble formation occurs.I dont know if it has sequencing vertical gel apparatus.
I hope this will help u. but i would like 2 remind u again to clean your plates very-very properly when u start ur work and also after u have done with ur work.
Also any gel part remaining on plates after u done ur work and its not clean properly can crack it. so beware!
i think u have problem with bubble formation during pouring of gel and not with sample loading, so there is no role of loading buffer or glycerol. If u had problem in loading samples into the wells, u can increase your loading buffer as it has glycerol which provides weight and thus lead ur sample to sit properly into the wells
Clean plate is necessary to avoid bubble appear.
I usually clean my plate with etanol. If u clean your plate with detergent or soap u must wash your plate properly.
practice make perfect..
Good luck
hai
wat pallavi said is right along with that add solution along side of glass plate wall slowly.it will avoid the bubble formation .
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