The PCR of my samples look great.
I used 0.5 uL of TruI enzyme and 2 uL of 10X buffer for digestion (1h @ 65°C) of PCR (432 bp).
Then I used 5 uL of dye for 22.5 uL of digested PCR, and run 12 uL of it in a 2.5% agarose gel.
The result is terrible. The other photo show the same tecniques carried out 4 months ago.
Why could happen this?
Extraction of DNA is not performed by legitimated handling or DNA sample is not in pure.
Dear Francesco,
If I was you I would first check all your calculations and the DNA quality and concentration you have before starting the experiment. DNA quality and concentration can be checked with Nanodrop. I assume that in your calculation you used 0.5 µL enzyme, 2 µL 10X buffer and 20 µL DNA in PCR-grade water (as you have 22.5 µL in the end?)? If having a 10X buffer you should dilute it like this: 9 µL water + 1 µL buffer (to get 1X).
As a next step check your consumables, are you always using PCR grade water and PCR grade pipette tips?
And as a last step, I would make new stock solutions (e.g. TAE for agarose gels etc) and renew the gel staining solution. Also if your fragment is approx. 450 bp in size you might go down with the agarose concentration a little bit, e.g. to 2% to get a better band separation.
I hope this was helpful!
Best
Eva
Another possibility might be that simply your TruI enzyme is not working anymore (because its expired, contaminated or was handled incorrectly)!
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