Is it possible that combs have accumulated DNA samples from previous loadings. Because for me it seems that every time we run a gel we first remove the comb just after casting and then only run our samples. Thus while the DNA samples run comb has already been removed. The only way of getting combs contaminated seems to be by touch and that to we touch it for cleaning and if we clean it then how it is still contaminated? In the attached gel I was doing a touchdown PCR to check for positives where I got only 2 positives in sample no.16 and C2 respectively.
L-Ladder(100 bp)
P-Positive Control
Number- Denotes my sample number
Amplicon size- 428 bp
But last 11 wells were not at all loaded but still they show a thin band just below the well (marked with red arrow). The size seems to be like total genomic DNA samples. The band is also visible in the ladder well and also in the loaded well samples. It is common to all the well whether loaded or not. So, it seems that it is from the comb!
Your comb would have to be extremely dirty. To get a band of that intensity it would have to have about 10 ng of DNA at the least sticking to the comb. I doubt that is your problem. Every gel is going to have some background due to buffer or uncast agarose. It looks to me like you should load about twice the amount of material that way your sample fluorescence is higher than your background fluorescence.
Your running buffer needs changing. Use fresh TAE, give your electrophoresis tanks a clean (be careful not to break the electrodes) and hopefully the problem should go away.
Being that high on the gel, next to the wells almost looks like genomic DNA. Question, are you using genomic DNA as your template and if so, how much are you using per reaction in ng? Sometimes you can see your background genomic DNA show up if you use way to much in your reaction. And as other's have said, clean everything thoroughly, switch out new buffer and use fresh agarose gels.
yes I have used genomic DNA as template whose concentration is as high as 300-500 ng/ul but this doesn't explain the appearance of these bands in the unloaded wells.
Dear Sahu,
The contamination bands may not be the results of contaminated combs. It is due to the fact that you are pipetting with air bubbles in the tips, which spills your loads from the loaded wells to the unloaded wells when the air bubble get out of the wells, or you are loading to much of the samples, creating also a spill over to other wells. You may not be loading your samples cleanly in the targeted wells. If your buffer in the tanks are dirty of loading dye after loading the samples, you have the answer. Secondly, are you recycling your gels? this may be another source of contamination.
Best regards
Dear Sahu,
This types of problem happen generally but not due to the comb's contamination. How it is possible that because prior to loading the samples we are removing the comb first and every time we are washing the combs before gel casting, even though we are using the fresh agarose gel to prepare so there is hardly any chance of the sample contamination. Secondly, the air bubbles may create the problem but only to the adjutant 1 or 2 wells. however, we can see the same intensity bands in all the empty wells, you must have seen like that. The same problems I have faced lot then I have analyzed the buffer several time. I used the very old buffer, some how old and very fresh and neatly cleaned. I came to know that in old buffer band intensity was suppose to like loaded samples whereas in some how old sample it was comparatively light but in the fresh buffer it was not at all. So conclusively we can say that in the old buffer there may be a lot of microbial contaminations, you must know that most of the buffers having the capacity to providing favourable conditions to grow the microbes. When we are applying the high voltage pulses to run the gel, same times microbial cells which are settled down to the wells may get burst and their DNA may get released and it run as usual.
I think you may get solved the problem and may get answered the question.
All the best
repeated use of tae buffer, gel or over run of gel may leads to such problems
I agree with dr. More and Dr. Ainsworth, your buffer might be quite old. change your buffer every 5 days and may be you solve out the problem!
All the best to you!
Hello Sahu,
one other possibility is increasing contamination in your loading dye. It looks to me like the high bands increase in intensity from the first row (where they are essentially absent) to the second row (where they are slight) to the third row (where they are clearly evident). Is there any chance that individual tips are used between multiple samples and the loading dye aliquots? On the other hand, if you didn't load anything at all into the wells after the second ladder in the third row, this wouldn't be a likely explanation.
Good luck,
Matt
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