I measured the DNA concentration using Nanodrop and made 10 μl total digest solution. I was wondering what would be the problem that I don't even get band for the uncut control sample?!
can you see your dna ladder?
If you add dna to an agarose gel and see nothing then some possibilities are
You forgot to add etbr to the gel
The bulbs in the transilluminator are off/broken.
you ran the gel in the wrong direction and the dna went off the end of the gel
your dna is,in fact,rna and is running as a smear in the gel
You did not load enough dna andit is not visible.
What you think is plasmid dna is actually genomic dna and is running as a smear on the gel
10 µl at what concentration? If you load e. g. 500 ng you should get a nice band. If this is not the case, you have either highly degraded DNA which does not occur with common plasmid prep protocols or a lot of RNA.
Either you purify your DNA and measure again, or if you just prepare minipreps don't care about the OD. Starting with 2ml culture depending on copy number, size, ... load just e.g. 1/20 and 1/50 on your gel.
Paul Rutland Thanks so much for your answer.
I've loaded the DNA ladder and used the same plasmid for the PCR that looks fine.
This time I will try to add more DNA into my digest mixture. Do you recommend any ng?
Uwe Borgmeyer Thank you Dr. Borgmeyer for your answer. I did PCR for the same plasmid and got bands. But what is the least DNA concentration you recommend for the digestion mixture?
As Paul mentioned, there may be technical problems.
Always load the same DNA ladder. With typical DNA ladder (e.g. Thermo 1kb plus) load 500 ng. The bands you should be able to visualize are typically in the range from 20 ng to 100 ng.
This is true for your plasmid. To be on the safe side cut approximately 500 ng.
Between 10-20 ng per band should be visible with etbr as stain. The amount you cut will depend on the number of cutsites/bands so if you expect 5 bands then 50-100ng bearing in mind that small bands intercalate less etbr so look fainter so 50bp bands may not be very obvious. The amount you can see also depends on the well size...smaller well means less dna is needed to see a band and distance run....bands diffuse as they run further so a small band may be visible after 10 minutes on the gel but becomes diffuse or invisible as it spreads out after 30-40 minutes running.
I am assuming that your dna is pure plasmid and not plasmid with rna or genomic dna when you meaured the amount present as the impurities will not give bands on restriction digestion
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