Agarose Leaks

[James Gardner]

In my biotechnology class we just finished running agarose gels, but the process was tedious. Manly because the gel trays we used were not enclosed on either side so we had to use tape to close the ends up so the agarose wouldn't leak. I asked my teacher if they made enclosed gel trays and he said they didn't. I find it hard to believe that this is the case. Is this true?

[admin]

Hello James,
they do make enclosed trays i hated the take too as the agarose sometimes leaks.

[redrivergyrl]

I have recently started running my own miniprep plasmids on an agarose gel. Initially, everything seemed fine, but lately, my bands are very smeared. The ladder has even started smearing. My PI and my labmates and I have troubleshooted for awhile about this problem (changing power boxes, different gel boxes, different reagents, different concentrations, etc.) They say they haven't seen anything consistenly like this. I re-tested plasmids that I initially tested and there was nothing wrong with them, but when I re-tested them, they also smeared. I am at a loss of what to try and do next - does this sound familiar to anyone? Any suggesetions? Help!

[esl]

I found that using autoclave tape to seal the gel tray is excellent and never leaks (if you position it right!), but using masking tape will definitely leak if the gel is hot, as the tape is not sticky enough.

Good luck!

[ashabo]

Originally Posted by James Gardner

In my biotechnology class we just finished running agarose gels, but the process was tedious. Manly because the gel trays we used were not enclosed on either side so we had to use tape to close the ends up so the agarose wouldn't leak. I asked my teacher if they made enclosed gel trays and he said they didn't. I find it hard to believe that this is the case. Is this true?

hi Jame!! you're professor have right because we can't prepare gel if our mould is enclosed, (it's question of flow of air!!) and to have an uniformely gel and no bublle in it. are you okay?

if no, you can ask me also.
best regard
Ayman

[ashabo]

Originally Posted by redrivergyrl

I have recently started running my own miniprep plasmids on an agarose gel. Initially, everything seemed fine, but lately, my bands are very smeared. The ladder has even started smearing. My PI and my labmates and I have troubleshooted for awhile about this problem (changing power boxes, different gel boxes, different reagents, different concentrations, etc.) They say they haven't seen anything consistenly like this. I re-tested plasmids that I initially tested and there was nothing wrong with them, but when I re-tested them, they also smeared. I am at a loss of what to try and do next - does this sound familiar to anyone? Any suggesetions? Help!

Hello friend,

in fact you're agarose Gel contain apparently ROSO3- residue which responsible to make saline bridge with the constituent of your plasmid on hand. on the other hand i think that you must work with fraction which diluted more and use detergent as well as tween 20, or other!! Finally, you must try migration on PAGE (poly acryl amid gel electrophorises). You have another choice, you can change your strategy and work with SDS-PAGE???

good luck and i'm ready to replay you if it's necessary!!

[Mathijs]

Originally Posted by redrivergyrl

I have recently started running my own miniprep plasmids on an agarose gel. Initially, everything seemed fine, but lately, my bands are very smeared. The ladder has even started smearing. My PI and my labmates and I have troubleshooted for awhile about this problem (changing power boxes, different gel boxes, different reagents, different concentrations, etc.) They say they haven't seen anything consistenly like this. I re-tested plasmids that I initially tested and there was nothing wrong with them, but when I re-tested them, they also smeared. I am at a loss of what to try and do next - does this sound familiar to anyone? Any suggesetions? Help!

Stupid question maybe, but are you sure that the agarose has completely dissolved? I sometimes notice that a very fast made gel that has not microwaved long enough has the same properties you describe. I would replace stock TBE/TAE as well.

Were did you elute/dissolve your plasmids in after the prep? Elution buffer contains EDTA, and chelates Mg2+, thereby reducing activity of DNAses. In ddH2O, samples can deteriorate more rapidly with increasing freeze-thaw cycles. This also greatly depends on the strain you isolated the plasmids from, as some strains do exhibit nuclease activity while others do not. What strain did you isolate your plasmids from? This does not explain smearing of the ladder though...