Hi Harrie,

I am not sure if I understand your question right. Could you give us some more infromation? Do you have trouble to decide which enzyme to use (is the plasmid sequence known?)? Or do you have technical trouble like incomplete digestion, smear etc? What is the plasmid in question? What is the purpose of the digestion - checking construction, verifying sequences etc.?

Best wishes,

Klaus

hi, Harrie.

could you give me some more information about this plasmid? I prefer use fermentas enzymes to cut plasmid, moreover, fastAP is recommened to add in mixture. 

The size should not cause the problem when you have high or medium copy number. Problems in digest could be DNA quality/purity. This can be solved by a clean up using columns from a PCR product and/or gel fragment purification kit.

What are you trying to accomplish by cutting the vectors?

As everybody said, we need more information to find out the possible issues. I have had good results with New England BioLabs enzymes.

If you have trouble digesting it, I could think of:

1 check your enzyme buffer (instead one-fits-all, you may use optimal buffer for your restriction enzyme; if you need to use two or more different ones, then try to cut first with the low-salt buffer, then ajust for the higher-salt buffer; or, precipitate and clean the DNA after the first restriction and use optimal for all digestions.

2. use fresh enzyme(s)

3. perhaps, you mean something different: if you have too many restriction sites and too many fragments, check for rare sites, perhaps with southern blots to make a better map of your potential insert.

The plasmids (10-16 Kb) are not difficult to digest by restriction enzymes. Although I cannot focus your question, I have some suggestion for you.

Beat wishes

thanks for all your suggestions.

The plasmid map is known. I do have trouble cutting the vector with restriction enzymes. The purpose of the digestion is cloning and confirmation of the cloning steps. I am now trying sodium acetate/ethanol precipitation to clean the DNA, followed by direct digestion with restriction enzymes. That seems to work. Leaving this cleaned batch of vector overnight at minus 20 C again leads to no digestion. Is it possible that secondary structure in DNA can inhibit enzymatic digestion?

Harrie

If you're using one enzyme to cut your plasmid, they will self-ligating after cutting them. You must check the MCS (multiple cloning site) on your plasmid and use two different enzymes to digest your plasmid. Also check that both enzymes are compatible with the temperature. Hope this helps!

Hi Harrie,

To solve your problem, you can put the frozen DNA in to 65℃ water bath for 5 min, followed the ice bath immediately. For more precise, untreated DNA is necessary to be used as a negative control.

Hi Qi Liu,

I was thinking about heating the DNA before digestion to get rid of secondary structure. I will try you suggestion. Thanks.