11 replies to this topic

[Prep!]

hi all.. i have an observation which is getting difficult to explain. I run an IEF all works fine i get desired results but the only hitch is that i get bands in the adjoining lanes too!!!
I am sure that it is not a spillage and i dosnt look like diffusion either as the band is not continuous. I have attacehd the stained gel and the blot for your reference. Please suggest.

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[Vini]

Pradeep Iyer, on Nov 23 2009, 06:30 PM, said:

hi all.. i have an observation which is getting difficult to explain. I run an IEF all works fine i get desired results but the only hitch is that i get bands in the adjoining lanes too!!!
I am sure that it is not a spillage and i dosnt look like diffusion either as the band is not continuous. I have attacehd the stained gel and the blot for your reference. Please suggest.

Hey PI, u sure that there's no spillage???.........looks like that to me   only wid ur sample or the marker also???

[Prep!]

No spillage i am very sure that is why i mentioned that clearly too!!!
I don run a marker in this gel!!!  
IT is glycosylated but i cant relate that to the banding pattern!!!

Totally lost!!!

[mdfenko]

even though you don't notice it, it does appear to be spillage. your sample may be crossing after you start the separation.

do you load blanks in the unused wells? doing this may prevent your problem.

[Prep!]

hey md... thanx for that suggestion.. no i don load any blanks..
will do that next time and get back with the results... ya it can be tat it is crossing over while focussing and not during loading!!...
by the way i load my sample at the start itself.. so it goes thru pre focussing as well as focussing voltages!!!

[Maddie]

I sometimes have this problem with agarose gels and to prevent is, I use more dye. The PCR product goes right into the bottom of the well. I'm not sure it would help in your case...it's just my 2 cents  thoughts.

[gogreen]

PI, It looks spillage to me too!! the bands are clearly the same!!

[Prep!]

Maddie, on Nov 24 2009, 11:00 PM, said:

I sometimes have this problem with agarose gels and to prevent is, I use more dye. The PCR product goes right into the bottom of the well. I'm not sure it would help in your case...it's just my 2 cents  thoughts.

hey maddie.. thanx for the reply.. i add enough dye to get till the bottom of the well.. anyways will try to add blanks as md suggested and will get back..  
hey gg.... i am damn sure its not spillage... the loading volume is 10 mcl and the well holding capacity is 40 mcl!!!! i have to be really not concentrating if i have to get a spillage!!!  

[gogreen]

Maybe u should be really concentrating in ur work PI

It is the same sample which is in all the 3 wells....Beats my sense to explain how this can happen other than a spill over!  

[Maddie]

Good luck, let us know how it worked   .

By the way, you sure it's not a problem with the film, right?

[eldon]

Maddie, on Nov 25 2009, 07:09 AM, said:

Good luck, let us know how it worked   .

By the way, you sure it's not a problem with the film, right?

classic spill over if there ever was such a thing.

or you are using precast poop gels that have expired, and rather than spillage from overfilling the well, it has seaped well-to-well.

i am not a fan of precast gels for many many many reasons.

Edited by eldon, 25 November 2009 - 03:54 PM.

[Prep!]

ya eldon.. tat is wat even i was thinking.. well to well seapage... so will add blanks in the adjescent lanes so address tat!!!
i am using hand cast gels only... thanx maddie.. by saturday eve here in india.. i l get back to u al...
and gg... hope u are right cause if u are not u might have to conc better in not letting things beat ur senses!!!