I digested my inserts with restriction enzymes and then purified them through columns. For the absorbance ratio (260/280), Nanodrop gave the value of 1.59, which indicates the presence of proteins (enzymes). However, the gel run showed the sharp bands. Now I need to proceed to the ligation reaction.
(i) Can restriction enzyme traces interfere with the ligation reaction?
(ii) Do I need to repurify it by the gel extraction method before proceeding to the ligation reaction?
If you have active restriction enzymes that could digest the ligated product, then you have a potential problem. If the restriction enzymes don't have a target in your DNA (before or after ligation) then it should not be a problem. Note that it might not necessarily be the enzyme, some RE buffers have BSA in them etc, so there might be other sources of protein.
Restriction enzymes can interfere if they are still active. Usually there will be an enzyme inactivation step through high heat (60 degrees or 80 degrees), but I'm not sure it won't affect. Normally I would ligase the product from the gel electrophoresis purified. The presence of proteins may not necessarily be proteins but their buffers, or if proteins are present, they may not be active.
As Michael J. Benedik and Hong Thai Le Nguyen answered the presence of active restriction enzymes could be an issue. Nevertheless, I believe you are overinterpreting the 260/280 ratio, I did (at least) hundreds of ligations in my life and never noticed the presence of active RE after column cleaning, especially since such usually includes washing with 70% ethanol, a good way to kill most enzymatic activity.
On the border subject, if ligation is designed properly RE can be added to the ligation reaction such as to lower the chances of self-ligation of the vector - this can be done by creating RE such that the resulting ligation results in a new linker that is not recognized by the original RE (i.e. using two different compatible-ends REs) or when ligating two PCR products and using methylation-dependent RE (usually DpnI) to remove the PCR template(s).
If you can, Heat denature your digested products before proceeding for ligation. This will eliminate RE activity and REs bound to DNA. This will improve ligation efficiency. Keep in mind, Only some RE can be denatured (check here: https://www.thermofisher.com/in/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/conventional-restriction-enzymes-thermo-scientific/reaction-conditions-for-restriction-enzymes.html).
Gel elution would be ideal, but keep in mind that the salts (guanidium thiocyanate) in the buffers used can mess with nanodrop readings. Check purified products on gel.
i) In most cases, the presence of residual restriction enzymes in your plasmid or insert solutions should not significantly interfere with the ligation reaction. Restriction enzymes are typically heat-inactivated or inactivated by other means during the purification process. However, it is good practice to ensure that the restriction enzymes are completely inactivated before proceeding with the ligation reaction. This can be achieved by following the recommended heat inactivation or inactivation protocol provided by the enzyme manufacturer.
(ii) Repurification of your DNA by gel extraction may not be necessary if you have already purified your inserts through columns and obtained sharp bands on the gel. Gel extraction can be an additional step to further purify the DNA if you suspect the presence of impurities or if you want to remove any residual proteins or enzymes. However, it is not always required if you have obtained satisfactory results from column purification and your DNA bands appear clean on the gel.
The presence of proteins (enzymes) indicated by the 260/280 absorbance ratio does not necessarily mean that the restriction enzymes are still active. The ratio can be influenced by other factors such as contaminants or impurities in the sample. Therefore, it is advisable to follow the recommended purification protocols for your specific application and assess the quality of your DNA samples based on the appearance of bands on the gel. If you are uncertain about the quality or purity of your DNA, you may consider performing additional purification steps such as gel extraction.
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