Using nanodrop to measure plasmid DNA concentration
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12 سال پیش
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Corrections and comment from Nanodrop:
Corrections and comment from Nanodrop:
NanoDrop does not "...pulse the sample with electrical current and measure the time it takes...". In fact, no electricity passes through the sample, only light, and the instrument measures light, not time or electricity. The buzzing noise you might be able to hear is the xenon flashlamp firing; the clicking sound is a solenoid under the arm that raises and lowers the arm, thereby changing the pathlength (if you crouch down and watch, you will be able to see the drop of sample being compressed and stretched during measurement). The concentration of the sample is calculated using the absorbance of the sample at 260 nm, the pathlength and a factor based on the extinction coefficient of DNA (Beer's law).
The essence of the idea is that the instrument measures the absorbance of the sample at 260 nm at different pathlengths, then does the calculations for you.
The NanoDrop does not "...eliminate the need for an ultraviolet lamp...": actually the xenon flashlamp it uses provides light in both the UV and visible light ranges. Xenon lamps don't need any warm up time (you might be used to having to wait for the deuterium lamp to warm up in some other spectrophotometers).
When you start using the instrument, you will be asked to put water on the pedestal -- I suggest 2 µL -- and the instrument will initialize. This is checking that the instrument is working properly. This won't detect dirty pedestals, although the initialization will fail if they are exceptionally dirty.
After you blank the instrument, the spectrum you will see will show no absorbance. This does not indicate whether your blank has any absorbance (to determine that, you would need to blank using water and measure your blank as if it were a sample, but this is seldom necessary). For your students, you rightly pointed out the importance of making sure that the blank is the same as the buffer in which your sample is suspended. Lots of people don't realize the importance of this.
We've never tried the blotting method of removing the sample from the pedestals: we always use more of a wiping motion. Don't be afraid of damaging the instrument as the pedestals are quartz fibers in stainless steel, so wiping with a lab tissue will never hurt them.
Another thing you will wish to change is that where you look at the spectrum and comment on the 230 nm absorbance. Proteins don't really absorb much there. As a general rule, carbohydrates absorb at 230 nm, DNA/RNA at 260 nm and protein at 280 nm. Contaminants such as guanidine (often from column based kits) will give absorbance at 230 nm, residual phenol (not normally used for plasmid preps) absorbs at 270 nm.
The advice I like to give students is to first look at the wavelength of the 230 nm trough and 260 nm peak. If either of these has moved to a different wavelength, you probably have a contaminant. Then look at the purity ratios: a poor 260/230 ratio means carbohydrate contamination or carryover from your kit, a poor 260/280 ratio indicates the presence of protein.
See http://www.nanodrop.com/ND1/NucleicAc...
NanoDrop does not "...pulse the sample with electrical current and measure the time it takes...". In fact, no electricity passes through the sample, only light, and the instrument measures light, not time or electricity. The buzzing noise you might be able to hear is the xenon flashlamp firing; the clicking sound is a solenoid under the arm that raises and lowers the arm, thereby changing the pathlength (if you crouch down and watch, you will be able to see the drop of sample being compressed and stretched during measurement). The concentration of the sample is calculated using the absorbance of the sample at 260 nm, the pathlength and a factor based on the extinction coefficient of DNA (Beer's law).
The essence of the idea is that the instrument measures the absorbance of the sample at 260 nm at different pathlengths, then does the calculations for you.
The NanoDrop does not "...eliminate the need for an ultraviolet lamp...": actually the xenon flashlamp it uses provides light in both the UV and visible light ranges. Xenon lamps don't need any warm up time (you might be used to having to wait for the deuterium lamp to warm up in some other spectrophotometers).
When you start using the instrument, you will be asked to put water on the pedestal -- I suggest 2 µL -- and the instrument will initialize. This is checking that the instrument is working properly. This won't detect dirty pedestals, although the initialization will fail if they are exceptionally dirty.
After you blank the instrument, the spectrum you will see will show no absorbance. This does not indicate whether your blank has any absorbance (to determine that, you would need to blank using water and measure your blank as if it were a sample, but this is seldom necessary). For your students, you rightly pointed out the importance of making sure that the blank is the same as the buffer in which your sample is suspended. Lots of people don't realize the importance of this.
We've never tried the blotting method of removing the sample from the pedestals: we always use more of a wiping motion. Don't be afraid of damaging the instrument as the pedestals are quartz fibers in stainless steel, so wiping with a lab tissue will never hurt them.
Another thing you will wish to change is that where you look at the spectrum and comment on the 230 nm absorbance. Proteins don't really absorb much there. As a general rule, carbohydrates absorb at 230 nm, DNA/RNA at 260 nm and protein at 280 nm. Contaminants such as guanidine (often from column based kits) will give absorbance at 230 nm, residual phenol (not normally used for plasmid preps) absorbs at 270 nm.
The advice I like to give students is to first look at the wavelength of the 230 nm trough and 260 nm peak. If either of these has moved to a different wavelength, you probably have a contaminant. Then look at the purity ratios: a poor 260/230 ratio means carbohydrate contamination or carryover from your kit, a poor 260/280 ratio indicates the presence of protein.
See http://www.nanodrop.com/ND1/NucleicAc...
12 سال پیش
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