In DNA/RNA Gel electrophoresis, which is the key factor, total quantity, or concentration, or volume, that may influence the lightness of bands?
Hi,
Actually, all the factors are involved because concentration=amount/volume. So all the factors are related and also involved in the intensity of your band on gel electrophoresis.
I hope this will help you.
If you look at your DNA-Ladder you should find the concentration they used for each band. With that you can choose the concentration for your DNA in your gel with your running conditions.
Usually 500ng of DNA/RNA would be enough to show you a visible band. I always use 500ng for RNA gel screening. The key factor is concentration. Stronger the band higher the concentration.
as others have said the well size and volume are important but with a small well and samples run about 5cm through the gel it should be possible to see 10-20ng dna in a single band. This amount varies with the intercalating dye used ( EtBr or SYBR or gelred etc). There is and effect also with the size of the dna and its structure....larger DS DNA looks brighter and is detected easier than small DS DNA or single stranded DNA both of which give weaker signals because they bind less dye
There are two aspects in your question one if you are looking for light and dark bands one important factor that influence gel electrophoresis is your running buffer TAE or TBE pH' along with quantity/concentration/volume of DNA.
secondly if you want to determine minimum concentration of your sample; load your sample in different concentration in wells then you can determine minimum concentration of sample that can be loaded into the wells and provide you desired results.
Paul Rutland a similar question on my part - in doing sequencing gels, would you have experience where running samples with large volume discrepancies affects the alignment of bands at the single-nucleotide resolution? I've been seeing some slight differences in migration of primer extension products (using Reverse Transcriptase, loading 30 uL) vs the Sanger Ladders (loading 2.5 uL); the primer extension products consistently migrate about 1/2 nt higher than the Sanger Ladders, which makes it not very straightforward to analyse, and also the primer extension bands appear to be narrower. I'm not sure if this is because of a volume difference, or perhaps I need to clean up my primer extension reaction with phenol/chloroform...
The Sanger Ladders, as per its kit, also has less formamide prior to loading (38% v/v) vs. my sample (47.5% v/v); I was wondering if this also makes a difference.
Benedict G Tan we should probablynot use Nan Zhous question for a different question.
Small differences are tricky and we should deal with them separately.
Are we talking flat gels or capillary electrophoresis. Which sequencing kit. What RT kit. Are you terminating the rt with dideoxy nucleotides or terminating at the end of the template. Salt concentration may also make a difference especially if it is capillary gel so it is probably necessary to look closely at the sample constitution
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