What are the disadvantages of activity stain ?

For the gel electrophoresis a 2 % agarose gel is made up (dimensions 20x20cm, 3-4mm thick). The gel is run for 4hrs at 85V. As running buffer 0.5x TBE is used.

A band pattern of 5-10 different bands (200-600bp) is expected depending on the sample. Some of the bands can be very similar in size and very close to each other. The gel image is not clear at all and the different bands cannot be distinguished.

Would high resolution agarose be a solution?

Hi, we sometimes get this weird stains on the agarose gels. We tried re-making TAE buffer and agarose from scratch (after some experiments, we were quite sure it was the buffer), we obtained a good gel (without those stains) but then the stains appeared again. Did anyone ever had that problem before? How can we fix it? Any idea what it is?

PS: On the right-hand side of the picture (where the stain is bigger) there was no sample loaded.

I ran RNA denaturing gel (1.2% agarose, 1X HEPES-EDTA buffer and 0.45M formaldehyde). Used 0.5 ug of RNA, 5 ul of loading dye (HEPES-EDTA, bromophenol blue) and 1ul of Etbr in the reaction. After addition the tubes were incubated at 70 C for 5 mins. 

Shouldn't all the bands be at the same position? I don't think the RNA has degraded because we can see distinct 28S and 18S bands as compared to RNA ladder. 

What may have caused this different size of band?

Is agarose gel with expired date (4 years stored in good conditions not opened yet) good for use?

We do routinely out agarose gel electrophoresis with Lithium borate buffer instead of using the classics TAE or TBE (reference: Brody, JR, Calhoun, ES, Gallmeier, E, Creavalle, TD, Kern, SE (2004): Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques 37: 598-602)

We face at the moment problems with Qiagen's QIAquick Gel Extraction Kit, having only very low yields (around 0.5 - 2 ng/uL) but "fat" bands, and we thought the LiBo buffer is a possible source of the problems. Qiagen had no idea when we asked them. And of course we'd like to avoid to go back to the classic buffers. We use standard gel chamber, low-melt agarose and use a NanoDrop for quantification.

Does anybody used kits for DNA gel extraction with this buffer and experienced problems or is it possible without any?

Thanks for any input.

I've been working with RNA agarose gel electrophoresis and having trouble to visualize positive RNA control samples(1ug) and RNA ladder(3ug). enclosed is the best gel image I've got (please ignore lane 3-5). The gel is stained in Gelstar(10X sensitivity to EtBr) in this case. I also tried EtBr post-electrophoresis staining and got even worse result. either case, the positive RNA and RNA ladder stained very poorly. and Ladder lanes are indiscernible. below is some other info how I ran the gel: 

1% agarose gel, with formaldehyde and reduction buffer from Northernmax

RNA ladder (3ug from invitrogen), RNA positive control (1ug) from Northernmax kit

3X volume of formaldehyde loading dye

65C water bath 15min prior to sample loading

5V/ cm electrophoresis

MOPS 1X buffer in DEPC H2O

Major question:

1\ what's the reason that 1ug/ug RNA control/ladder wont give clear bands?

Other questions:

2\ the 1ug RNA control in lane 1, it seems intact and not smeared. does that indication no degradation?

3\ I used the agarose provided in the northernmax kit to run RNA gel. Can I use regular agarose (used for DNA) to run RNA gels?

I tried to run DNA extracts on an 1% agarose gel, at 100V for 30 minutes (it's a small gell), however I don't get any visible bands at the gel. I believe I have DNA as this was shown after measuring the samples on Nanodrop after the extraction.

I added both 3 and 6 uL of template unamplified DNA extract (as the DNA concentration was low), but still I don't get any bands.

Could it be that I need to increase the % of agarose, as the fragment is expected to have a large size, being a DNA extract and not an amplified fragment after PCR?

Thank you very much.

Is it normal that when an RNA agarose electrophoresis is done the ethidium bromide runs up the well? I mean, it goes to the negative electrode and I dont know if this is normal

Hello,

In my dept. we were making 15cmX10cm gel previously with the help of "Agarose D-1 Medium EEO" and everything was working fine from handling to getting bands of 100-1000bp.

However, now we are preparing 15cmX10cm gel from 1.6gm NuSieve Agarose (GTG) and 1.6gm Agarose (Ultra Pure) in 80 ml 1X Agarose Gel Buffer (pH8.6). These days we are getting trouble in handling gel. This gel is not tough enough till analyzing gel result. After finishing with electrophoresis part (at 100V for 1hr 30 minutes) when we are trying to put gel in UV unit we get small small pieces of gels within few minutes.

Kindly tell us what to do in this situation? If anybody knows protocol and handling techniques for this NuSieve Agarose (GTG) gel please share.