I extracted plasmid from the e.coli k12 strain using qigen plasmid midi prep. However, the nanodrop readings of the final product are consistently low (low yield); therefore, I adjusted the incubation time for the culture used for extraction so that my od600 reading was 0.94. I also used new isopropanol and LyseBlue to optimize the lysis of bacteria. However, I still get a very low plasmid yield on Nanodrop and a high one based on gel electrophoresis. This summer, the kit was working perfectly okay with similar-sized plasmid. Also the glycerol stock has never been refrozen, and bacteria grow well on agar plates. Does anyone know the reason for such low values, and is there any solution to solve this problem?
Try to quantify using the UV-VIS spectrophotometer.
As all the nanodrop instruments are not calibrated to quantify the plasmids.
"Gel electrophoresis indicates that plasmid is present and is in high amounts in the final product. However, samples collected during the process do not produce any bands" - how does that look like? It seems to me that some of the Prep components have gone bad. Have you tried the alkaline lysis protocol or other plasmid isolation kits?
Gel electrophoresis is not really a quantitative method, so you can see a bright band with DNA concentration being somewhat low. Plasmid size and copy number should be taken into consideration. There is a lot that could go wrong with plasmid isolation (including antibiotic selection). At this point you could concentrate the samples you have by precipitation.
I always got better yields when I lowered the pellet:buffer ratio – so instead spinning 3mL of culture in one Epp tube I used three tubes, 1mL each and after the lysis+precipitation procedure I added the clear supernatant onto one column. That way there is a greater chance for a complete reaction (lysis and chromosome precipitation). Sometimes if there’s too much cellular debris the plasmid (especially the bigger ones) gets precipitated with it. You can also rinse the bacterial pellet with TE after spinning the culture. It gets rid of the media components and makes the cells less “sticky” (they react better with the lysis buffer).
I would look at doing a few things that I have seen in situations like this.
1. Clean the nanodrop sensor. Over time the sensor gets dirty and does not determine conc correctly. the ratios will still look good but conc is low. There is a special kit that you can purchase from Nanodrop that will work. You can use a small amount of ethanol and allow to sit on the sensor for 30 minutes then wipe off. This will help to solubilize residues. You can rule out calibration as nanodrop uses a xenon bulb that lasts a min of 30,000 measurements per Fisher
2. Check on UV-Vis absorbance and do the math
The concentration of DNA in a sample can be calculated using the formula:
C=(A260×CF)/l
where:
- C is the concentration of DNA in μg/mL,
- A260 is the absorbance at 260 nm,
- CF is the conversion factor (50 μg/mL for double-stranded DNA),
- l is the pathlength of the cuvette in centimeters (usually 1 cm).
3. if you have really bright bands on gel try doing gel extraction followed by EtOH precipitation and quantify
"Gel electrophoresis indicates that plasmid is present and is in high amounts in the final product" how much did you load on your gel? if yopu put 10ul at 10ng this make 100ng on the gel could make a nice band...
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