8 replies to this topic

[whoknows]

can someone help me?

lately i've been running alot of bad gels. It seems i can't run a gel for some reason and i'm pretty embarrassed.

the bands are all smeared and sometimes bands come out jaggedy as well.

I'm pretty careful to visually check to see that there are no particulates still undesolved in the agarose solution as it comes out of the microwave.

i typically don't wait that long before pipetting in ethidium bromide and pour the gel right away.

i've been letting the gel cool in refrigerator.

my question is whether quick cooling causes the smearing? do i always have to let it cool down gently at room temp? is that my problem?

is there a fundamental difference in terms of the polymerization and microscopic structure of the gel when it's left to cool slowly at RT rather than quickly cooled in refrig or even freezer?

Edited by whoknows, 21 October 2009 - 02:26 AM.

[hobglobin]

whoknows, on Oct 21 2009, 11:27 AM, said:

can someone help me?

lately i've been running alot of bad gels. It seems i can't run a gel for some reason and i'm pretty embarrassed.

the bands are all smeared and sometimes bands come out jaggedy as well.

I'm pretty careful to visually check to see that there are no particulates still undesolved in the agarose solution as it comes out of the microwave.

i typically don't wait that long before pipetting in ethidium bromide and pour the gel right away.

i've been letting the gel cool in refrigerator.

my question is whether quick cooling causes the smearing? do i always have to let it cool down gently at room temp? is that my problem?

is there a fundamental difference in terms of the polymerization and microscopic structure of the gel when it's left to cool slowly at RT rather than quickly cooled in refrig or even freezer?

Mostly this is due to overused buffer in the gel chamber, replace it and try out again...About fridge I'm not sure (never did it), but normally the longer you leave the gel too harden the better it is, i.e. better 2h than half an hour...the faster hardening in the fridge might also influence the gel structure, i.e. less order, more distortion. But also this you can easily compare to a "normal" prepared gel...

[whoknows]

i prepared the buffer fresh from the 50x lab stock that everyone uses. so i'm pretty sure it isn't the buffer.

hobglobin, on Oct 21 2009, 04:28 AM, said:

whoknows, on Oct 21 2009, 11:27 AM, said:

can someone help me?

lately i've been running alot of bad gels. It seems i can't run a gel for some reason and i'm pretty embarrassed.

the bands are all smeared and sometimes bands come out jaggedy as well.

I'm pretty careful to visually check to see that there are no particulates still undesolved in the agarose solution as it comes out of the microwave.

i typically don't wait that long before pipetting in ethidium bromide and pour the gel right away.

i've been letting the gel cool in refrigerator.

my question is whether quick cooling causes the smearing? do i always have to let it cool down gently at room temp? is that my problem?

is there a fundamental difference in terms of the polymerization and microscopic structure of the gel when it's left to cool slowly at RT rather than quickly cooled in refrig or even freezer?

Mostly this is due to overused buffer in the gel chamber, replace it and try out again...About fridge I'm not sure (never did it), but normally the longer you leave the gel too harden the better it is, i.e. better 2h than half an hour...the faster hardening in the fridge might also influence the gel structure, i.e. less order, more distortion. But also this you can easily compare to a "normal" prepared gel...

[HomeBrew]

I could see a situation whereby the outer surfaces of the gel solidify quickly in the fridge, then act as an insulator so the middle of the gel doesn't harden as much in the amount of time allowed -- this would likely lead to smearing.  All theoretically, of course...

If your smearing problems started after you started hardening your gels in the refridgerator, there's likely a link -- don't do that anymore.  I've never done that, nor heard of anyone who does -- if we're in a hurry, we'll cool the agarose down in the flask by swirling it while running cold water from the tap on it.  Although this works without problems, I don't do it often -- if I'm that up against the clock, I haven't planned my experiment (or my day) out well...

[swanny]

HomeBrew, on Oct 22 2009, 07:43 AM, said:

if we're in a hurry, we'll cool the agarose down in the flask by swirling it while running cold water from the tap on it.

I typically make my gels up in 1/2 the required volume of buffer, then add the remaining buffer once the agarose has melted, which instantly drops the temp to something to ~60C.

[HomeBrew]

That's a good idea, swanny -- I'd never heard of that one, either...

[gebirgsziege]

I can support HomeBrews theory.....once tried to solify the gel in the refridgerator (bad planned day )  I had to repeat the gel the next day....was very messy.

But another reason for smeared bands can be too high voltages (more than 10V/cm) used for running the gel, esp. if you run your gel longer than ten min. The buffer gets very hot and the resolution of the gel gets very bad especially if running more gels in the same tank without changing buffer (experience from another bad planned day).

Swanny will try this the next time I run too many exp at one time  

Edited by gebirgsziege, 21 October 2009 - 10:17 PM.

[Adrian K]

As HomeBrew mentioned, the culprit is the fridge.

I melt my gel almost like the way swanny did. However, I add roughly 25% of water each time and wait beside microwave till i see it boiling. The final 25% was added without microwave, then I did what HomeBrew mention by swirling inside a pool of water. Usually I will add little ice to make it more "cooler" (mind you, i'm in tropical country, water is slightly warm). I will swirl roughly for 1.5 minute but never longer than that, and pour the gel directly. The gel will solidify roughly 20-40min and ready to run...I never had any issue.

BTW for my gel I use 1.5% agarose (vivantis), and run for 140V for 30 minutes, on a quite dirty buffer (only replace perhaps one in a month). Don't really see much of smearing effect (only very little) or any issues.

[mdfenko]

perhaps whoknows used low melt agarose. then the gel may have melted during the run.