Can we use regular Taq for cloning construction? Do we still need high fidelity enzymes?

When choosing domestically produced high fidelity Taq, the main factors to consider are whether it has hot start, whether it has calibration activity, fidelity data in the instruction manual, and typical amplification length. Small packaging of 250U/500U is more suitable for laboratories with small cloning volumes. Inquiry should not only be based on the unit price of the large packaging, but also on the compatibility of the A solution and buffer solution. If expression vectors are to be used in the future, it is recommended to prioritize high fidelity sequencing to confirm individual clones and reduce the risk of downstream expression abnormalities caused by mutations.

The choice between high-fidelity and regular Taq is a balance between enzyme cost and sequencing screening cost. Domestic high-fidelity enzymes are usually sufficient for routine CDS amplification and are more affordable than imported ones, but do not pursue only the highest fidelity multiplier. First use a 1.5 kb template for a small-scale test to check yield, background, and A-tailing efficiency. False positives or nonspecific amplification are related to the enzyme, primers, and cycle number, and cannot be judged by brand alone; it is best to evaluate them together with sequencing results.

The safe route for CDS to T vector sequencing is to amplify with high fidelity enzymes, add A tail, and then clone TA. Ordinary Taq comes with a convenient A tail, but the risk of 1.5kb amplification mismatch is not low, and the subsequent screening cost will increase. If using regular Taq, it is recommended to limit it to 25-28 cycles, select at least 8-10 monoclonal antibodies for full-length sequencing, and focus on the CDS ends and key structural domains, rather than just enzyme digestion identification.

If you insist on using regular Taq, it is recommended to select more than 8 clones from the same batch, first perform enzyme digestion or colony PCR preliminary screening, and then send them for bidirectional full-length sequencing. It is not advisable to select only 2-3 positive clones for sequencing. If the product of high fidelity enzyme amplification is flat end, Taq plus A or flat end cloning kit can be used to avoid low ligation efficiency caused by incomplete A tail. The low-cost approach is to use regular Taq for initial screening and high fidelity enzyme amplification for the correct fragment.