Expression Technologies Inc.
FAQs about DNA ladders/DNA markers
FAQ 1. How do you determine the size and quantity of a DNA sample with these DNA ladders/DNA markers?
Load a few microliters of the DNA sample together with a few microliters of a chosen DNA ladder on a gel containing DNA dye such as ethidium bromide. Run the gel with a DC power supplier. The DNA fragments in the DNA sample and in the DNA ladder will be separated on the gel based on their sizes. Smaller DNA fragments migrate farther than the larger ones. DNA fragments will be visible as DNA bands under UV light with ethidium bromide.
Compare the relative migrated position of the DNA fragment of the DNA sample to the relative position of the DNA fragments of the DNA ladder to determine the size of the DNA fragment of the DNA sample. For example, if the testing DNA fragment migrates to the same position as the 1 kb DNA band of the DNA ladder, the testing DNA fragment is 1 kb.
Compare the relative staining intensity of the DNA fragment of the DNA sample to the relative staining intensities of the DNA fragments of the DNA ladder to determine the quantity of the DNA fragment of the DNA sample. For example, if the testing DNA fragment has the same staining intensity as that of the 1 kb DNA band of the QunatDNA50-15KTM DNA ladder at 5 ul loading, the testing DNA fragment is 50 ng.
A DNA sample may contain multiple DNA fragments. Many DNA samples may be run on one gel.
We normally load different volumes (e.g. 10 ul and 2 ul) of a DNA ladder in the first and last lanes of a gel to orient the gel and to increase the quantity range. With these loadings, the quantity range can be increased from 20 times to 100 times with our DNA ladders.
The recommended loading volume for these DNA ladder/DNA markers is 5 ul. However, other volumes may be loaded depending on gel size and quantity of DNA sample to be determined. For large gels with big loading wells, 10 to 20 ul may be loaded. For mini gels with small wells, 1 to 2 ul may be loaded. Most agarose gel can detect 1 ng DNA band (Molecular Cloning, Sambrook et al). Under most electrophoresis conditions, all DNA bands in the QuantDNATM50-15k DNA standard can be detected with ethidium bromide staining by a regular digital camera, even when 2 ul is loaded. 50 and 100 bp bands may not be visible when 1 ul or 2 ul is loaded because of small DNA fragment diffusion on low concentration gels (<2%). Different loading volumes can increase the quantity range of the DNA ladders/DNA markers.
All of our DNA ladders/DNA markers have gradients of quantity and can be used to accurately estimate DNA quantity regardless of the size of the testing DNA. These DNA ladders/DNA markers are prepared by CsCl double banding. Extensive effort has been made to ensure its quantity accuracy. Quantification on the gel can save significant amounts of time and labor compared to UV reading, especially when many DNA samples are tested. Quantification using our DNA ladders/DNA markers is more accurate than UV reading. This is simply because testing DNA samples are often contaminated with impurities including but not limited to RNA, protein, oligos, or nucleotides. UV reading results will include these contaminants and thus over-estimate the DNA quantity.
Quantity accuracy also depends on the quantity interval of adjacent gradient bands. For example, if the staining intensity of a DNA fragment is between 700 bp and 800 bp bands at 10 ul loading volume, its quantity is about 75 ng. Its quantity should not be less than 70 ng or more than 80 ng. Therefore this quantity determination accuracy is + 5 ng.
Yes. Make sure the gel is evenly stained when you compare different DNA bands on the gel. If the gel is not evenly stained or if ethidium bromide is running off the bottom of the gel, the quantity will not be accurate. Our standards may be used to calibrate a density scanner.
Yes. When you load this standard on a buffer-less gel, make sure to add some water in the same well to fill half of the well. Otherwise the DNA bands cannot be well separated.
FAQ 6. Why are low or high molecular weight (MW) DNA bands of these DNA ladders/DNA markers sometimes not resolved or detected?
The figures of these DNA ladders/DNA markers are prepared on large gels. When smaller gels are used, only low percentage gels (≤0.8%) and high percentage gels (≥2%) will separate and detect high MW bands and low MW bands respectively. 50 and 100 bp bands may not be visible when 1 ul or 2 ul is loaded because of small DNA fragment diffusion during electrophoresis.
Yes. Each DNA fragment of these DNA ladders/DNA markers has 4 nucleotide 5' overhangs at both ends. These 5' overhangs may be labeled by T4 polynucleotide kinase with γ-32P-ATP. They may also be labeled by a Klenow fragment with α-32P-dATP since all overhangs may be filled in with dATP plus dTTP, dCTP and dGTP.
Knowing the quantity of a DNA sample is important for many biochemical studies.
DNA fragments generated from PCR or plasmids will be used in other biochemical analysis. Their quantities can facilitate these studies.
In DNA sequencing, quantities larger or smaller than the required amounts will result in shorter sequence reading.
In DNA cloning or subcloning, incorrect amounts of vector and insert DNA will lead to less efficient cloning. It may also produce wrong constructs in some blunt end cloning.
In construct analysis, quantities will determine the molar ratio of the vector and insert DNA. If the 1:1 molar ratio is correct, other ratios indicate incorrect constructs.
If both size and quantity are required, direct quantification on the gel is much more efficient than quantity determination by UV reading, especially when the sample size is large.