I have a 3.1 kb plasmid and I performed a digestion with the XmnI enzyme (37°C x 15 min and the enzyme was inactivated at 65°C x 20 min). When performing agarose gel electrophoresis, the size of the plasmid is approximately 6.2 kb. What could be the possible reasons that explain the growth in the size of the plasmid? Is the restriction enzyme working correctly?
Thank you for your answers.
The plasmid was coiled and supercoiled into a tight shere in its intact form so moves easily through the agarose gel but after restriction digestion the shape is linear so mich longer and spinning due to heat input from the gel so appears to be a much larger sphere and moves slower than the original uncut plasmid
Hello Kenia Chávez Ramos
If the desired size of the plasmid is 3.1 kb and you obtained a band over 6 kb, then there must be some problem with the plasmid construction. It might be possible that the insert was ligated multiple times in the vector. The linear plasmid should give 3.1 kb band (if it digested with a single cut restriction enzyme).
To find out the problem, you can perform control restriction digestions.
To check the enzyme efficiency, run a control linearization with another plasmid having a single XmnI restriction site.
To check the plasmid size, perform a control linearization reaction with another single cut restriction enzyme and compare the bands.
Best wishes.
Hello,
I don't see a direct reason for this problem with simple digestion, why the size changes and gives double the expected size.
Either, it is not your target plasmid that has been digested, check its size without digestion.
Either, your size marker is not correct, which resulted in a false size.
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