6 replies to this topic

[Bassaml7]

http://imageshack.us...newgellll2.png/


What you see in the link above is an image for a 2% agarose gel in 0.1X TBE buffer run for 30 minutes at 23 V/cm. The separation distance is about 15 cm. The bands are for a ladder (300,280,260,240,220,200,180,160,140,120,100,80,60,40,20). The 20 bp moved out of the gel, but that's ok. The problem is about these two lanes on the right with the "diagonal" bands. The other lanes are ok and I think it's an achievement to separate such small and closely spaced bands using just 2% agarose within just 30 minutes. However the last few lanes on the right always annoy me with that appearence. Also it's not just about the shape of the bands. As you can see there is a massive decrease in mobility. It appears that these bands move slower than the others on the left lanes, especially the small bands (please compare the bright 100 bp bands). It happens over and over. I make fresh buffer everytime because I know I am using a highly diluted buffer that can get consumed quickly. The current is just 65-70 mA and I think the temperature of the gel stays below 40 C during the run. By the way I pour the gel in the lower right corner of the tray. I pour it very hot so that it does not solidify. Yet I still get these diagonal irregular and fuzzy bands. If it's about heat then why are the left edge lanes never affected? I have loaded equal volumes in the wells. The loaded volume is 1/4 the capacity of the well and there is about 30-60 ng per band. It's very strange. I always get this same problem with last few lanes on the right side (always the right). Just why doesn't it happen on the left side? is it because I pour the gel in the right side? but it's not a concentrated gel (just 2%) and I pour it while it's very hot. So I don't think there's something physically wrong with the matrix of the gel.

Any help would be appreciated.
Thank you

Edited by Bassaml7, 16 May 2011 - 06:56 AM.

[phage434]

I'd guess it might be your gel box and the arrangement of the electrodes in the box.  Have you tried a different one, or examined where the electrodes are in the one you have?

[Bassaml7]

phage434, on 16 May 2011 - 07:30 AM, said:

I'd guess it might be your gel box and the arrangement of the electrodes in the box.  Have you tried a different one, or examined where the electrodes are in the one you have?

Thank you for your reply. In fact I noticed the aberrant shape of these bands does not appear under classical electrophoresis conditions (i.e. 0.5-1X TBE and 5-10V/cm). In addition, the larger the separation distance the more pronounced the difference in mobility between regular and irregular lanes. As you may have noticed the 100 bp bands appears in a position as if it were a 120 bp band when comparing with the left lanes. This causes a serious problem in genotype determination. I am developing a RFLP-PCR assay in which the difference in length between the indicative fragments is about 15-25 bp so I need accurate sizing. My fragments are also as small as those in the ladder. I am trying to optimize electrophoresis conditions and I think it was good to have everything separated within 30 minutes. I just got stuck with this silly issue. I haven't tried another tank. Maybe I will do later, but I don't think it has to do with the tank.

Thanks for your input

[chromatin]

Bassaml7, on 16 May 2011 - 07:53 AM, said:

phage434, on 16 May 2011 - 07:30 AM, said:

I'd guess it might be your gel box and the arrangement of the electrodes in the box.  Have you tried a different one, or examined where the electrodes are in the one you have?

Thank you for your reply. In fact I noticed the aberrant shape of these bands does not appear under classical electrophoresis conditions (i.e. 0.5-1X TBE and 5-10V/cm). In addition, the larger the separation distance the more pronounced the difference in mobility between regular and irregular lanes. As you may have noticed the 100 bp bands appears in a position as if it were a 120 bp band when comparing with the left lanes. This causes a serious problem in genotype determination. I am developing a RFLP-PCR assay in which the difference in length between the indicative fragments is about 15-25 bp so I need accurate sizing. My fragments are also as small as those in the ladder. I am trying to optimize electrophoresis conditions and I think it was good to have everything separated within 30 minutes. I just got stuck with this silly issue. I haven't tried another tank. Maybe I will do later, but I don't think it has to do with the tank.

Thanks for your input

I totally agree with phage434. I think you haver your own answer. Why do not you use 0.5x TBE. I have never heard anyone using 0.1x TBE. Looks like some pH, current, etc differences are occurring during the run. And when you use higher capacity buffer i.e. 0.5x-1xTBE, problem disappears. I do not know why you are insisting on using 0.1x TBE buffer. Also, if you are looking ro 15-25 bp difference why do not you sue higher percentage agarose such as 4%. Let us know how it goes. Good luck!

[Bassaml7]

chromatin, on 18 May 2011 - 02:10 PM, said:

Bassaml7, on 16 May 2011 - 07:53 AM, said:

phage434, on 16 May 2011 - 07:30 AM, said:

I'd guess it might be your gel box and the arrangement of the electrodes in the box.  Have you tried a different one, or examined where the electrodes are in the one you have?

Thank you for your reply. In fact I noticed the aberrant shape of these bands does not appear under classical electrophoresis conditions (i.e. 0.5-1X TBE and 5-10V/cm). In addition, the larger the separation distance the more pronounced the difference in mobility between regular and irregular lanes. As you may have noticed the 100 bp bands appears in a position as if it were a 120 bp band when comparing with the left lanes. This causes a serious problem in genotype determination. I am developing a RFLP-PCR assay in which the difference in length between the indicative fragments is about 15-25 bp so I need accurate sizing. My fragments are also as small as those in the ladder. I am trying to optimize electrophoresis conditions and I think it was good to have everything separated within 30 minutes. I just got stuck with this silly issue. I haven't tried another tank. Maybe I will do later, but I don't think it has to do with the tank.

Thanks for your input

I totally agree with phage434. I think you haver your own answer. Why do not you use 0.5x TBE. I have never heard anyone using 0.1x TBE. Looks like some pH, current, etc differences are occurring during the run. And when you use higher capacity buffer i.e. 0.5x-1xTBE, problem disappears. I do not know why you are insisting on using 0.1x TBE buffer. Also, if you are looking ro 15-25 bp difference why do not you sue higher percentage agarose such as 4%. Let us know how it goes. Good luck!

Thank you for your reply. In fact I am insisting on decreasing the buffer concentration in order to decrease conductivity so that I can apply higher voltage to separate the fragments faster without too much heat generation. High conductivity means high current and therefore excessive heat generation. I didn't want to go as high as 4% agarose because 4% agarose can solidify while you pour them in the tray and bands can look really nasty then. I eventually doubled TBE concentration and used 0.2X TBE and reduced the electric field to 14 V/cm which is higher than the conventional value (5-8 V/cm). It works well for me. I found that running at high voltages can enable intermediate concentration of agarose (1.5-2.5%) to separate small fragments, and within shorter time. However, that's on the expense of markedly higher separation distances so a big gel must be used. This method saves TBE too. However it's not suitable for long electrophoresis times. Anyway I think that's better than dealing with the 4-5% agarose gels which can be annoying sometimes.

Edited by Bassaml7, 21 May 2011 - 10:52 PM.

[phage434]

If you are trying to separate short fragments, then Nusieve 3:1 or Metaphor agarose is dramatically better than plain agarose.

[Bassaml7]

phage434, on 22 May 2011 - 05:41 AM, said:

If you are trying to separate short fragments, then Nusieve 3:1 or Metaphor agarose is dramatically better than plain agarose.

Yes that's a feasible option but I just want to show that standard agaroses can do the trick provided we use optimal electrophoresis conditions. It's a part of the subject of my research.

Do you have any idea how Metaphor or NuSieve differ from the standard agarose? I mean is it just that they are more purified and exhibit lower electroosmotic  flow or there's something different on the molecular level? I know they have an intermediate melting temperature so that implies a different chemical composition.

Edited by Bassaml7, 22 May 2011 - 07:02 AM.