Hello everyone,
I know it is a very basic question. Whenever I extract plasmid by alkaline lysis method and take nanodrop reading, it comes around 03-05ug/ul. Nanodrop peak is at exactly 260nm, 260/280 and 260/230 all within recommended range. But when I use it for digestion (e.g. 2-3ug) its intensity doesn't look like this. It seems faint as shown in attached gel picture.
Any suggestion or idea about this problem.
Hi Aditya. From gel picture it seems to be quite good yield of plasmid DNA. As you have mentioned that you get nanodrop reading of 3-5ug/uL. For digestion as you have mentioned 2-3ug DNA is used, means you are using 1 uL or less than that for digestion. Further if the digestion reaction volume is 10 uL or 20 uL, your DNA must be getting diluted by 10 or 20 folds in the reaction tube. From this if you load 5 uL or 10 uL (half of the digestion reaction), will give you 1 to 1.5ug digested DNA that too, split into two different fragment which would again reduce intensity as compared to original concentrated undiluted and undigested DNA. Hence it would be better to compare- in one lane loading your undigested DNA diluted to lets say 10 uL and loading five microliter of it, along with digested one. Still you will see that undigested in having more intensity than digested DNA as digested DNA fluorescence is divided into two fragments.
I usually load all the sample after digestion if its 20ul reaction. So, in the gel picture, theoretically, it's around 3-5ug/ul as I loaded all the sample in one well. But intensity doesn't tell the same story. As last time, I used 10ug of plasmid (as per Nanodrop readings), and after digestion, I loaded all the sample, but it was faint.
Do NanoDrop need calibration?
if your samples are diluted before measuring on the nanodrop check that the dilution factor is correct on the nanodrop settings, Also be careful that the solution that you use to zero the nanodrop is exactly the same as what your dna is dissolved/diluted in, eg you should not zero with water for samples diluted in TE. Nanodrop optics do need cleaning with dilute hydrochloric acid occasionally as protein and dna build up on the optical cell but good zeroing will account for much of this effect but it may be a good idea to measure a sample on another labs nanodrop if one is available
- Well said Paul !! Users of Nano drops tend to forget that about 1 x per month you should subject each pedestal to 5 min with 2ul of 1M HCL to hydrolyse contaminants that render readings in accurate and in consistent
- Commercial cleaners for both pH electrodes and nano drops tend to comprise pepsin and HCL (not together)
- Cleaning ad hoc when problems arise or 1 x per month routinely will lead to optimal and consistent output from your nano drop
- There is something sometimes more fundamental however: Nanodrop readings at 260nM actually measure organic residues which include phenolics like phenol itself or Trizol ( by resonance excitation of delocalised pi electrons in benzene rings). Thus, if your sample contai8ns trace amounts of substances that contribute to this reading your concentration by Spec or nanodrop will tend to be overestimated
- Thus, regardless of nanodrop contamination/performance issues, in situations like these I have always advised users to run 200-500ng of DNA estimated by Nanodrop to make sure it really is at the expected concentration relative to a known mass of ladder
- Once verified 500ng is a suitable amount to digest and run on a gel
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