Frequently Asked Questions

Ansa offers direct, contiguous synthesis of double stranded DNA up to 900 bp, and clonal DNA up to 50 kb. Our innovative technology enables us to deliver complex DNA sequences that are often challenging or impossible for other providers to synthesize. At the same time, our streamlined process handles more common sequences with ease, offering speed and unmatched reliability.

Every Ansa clonal assembly is sequence-verified using PacBio HiFi sequencing, a technology that can read the entire plasmid end-to-end with very high single-molecule accuracy. This allows us to confirm that the final DNA sequence matches what was ordered.

To pass QC, each plasmid must meet minimum material requirements and produce sequencing data that resolves into a single, unambiguous consensus sequence. This consensus must match the requested sequence. Because long homopolymer sequences are inherently both difficult to manufacture and to QC, we apply well-defined tolerance rules for those regions to ensure accuracy while accounting for known technical sequencing limits.

For additional rigor, we also evaluate several internal sequencing quality metrics. The target insert and the plasmid backbone are assessed independently but held to the same standards. The entire plasmid must pass QC, not just the insert. Sequencing coverage must be sufficient, continuous across the full sequence, and uniform. Beyond calculating the consensus, we also detect minor-frequency sequence variants (SNPS and indels) as well as structural variants to ensure the plasmid population is predominantly single, correctly sized molecules rather than mixed species or plasmid dimers.

Together, these checks allow us to confidently identify plasmids that are both sequence-accurate and structurally correct, and to clearly explain why a sample does or does not pass QC.

“Percent perfect” is the proportion of long read sequencing reads from an Ansa DNA Fragment that align to the expected sequence with no errors. This number is an estimate of the proportion of DNA molecules within your sample that perfectly match the expected sequence. Percent perfect is on a scale of 0-100% and is reported only for Ansa DNA Fragments; Ansa Clonal DNA originates from a single clone (E. coli colony).

Why is it important?

At Ansa, we want to be transparent and provide you all the necessary information needed to ensure you are successful in using our products. The percent perfect estimate is helpful for choosing a cloning strategy and, in particular, deciding  how many colonies you’ll have to pick to find a perfect clone containing your sequence. 

Percent perfect of a particular sample, as opposed to the more commonly  reported “average number of errors per bp” for a vendor, is a much more useful metric for downstream applications. Highlighting this distinction between error rate and percent perfect is important for the following reasons:

1. For many complex sequences, percent perfect (associated average error rate) varies from sample to sample, regardless of the vendor and technology used. 
2. Especially for complex sequences, the error rate can be non-uniform within a sequence, spiking in regions of high complexity. Using average error rate can therefore be misleading, implying a higher proportion of perfect DNA molecules than truly exists in the sample.
3. The percent perfect informs the number of colonies you’ll have to screen to isolate a perfect clone of the DNA Fragment with high probability.

How to use percent perfect to plan your experiment

In general, the higher the percent perfect score, the fewer clones need to be picked to have a higher likelihood of identifying a clone that contains the exact sequence. For example, to have a > 95% chance of identifying a perfect clone with a 60% perfect DNA Fragment, you’d have to screen 4 colonies, whereas to have the same chance of identifying a perfect clone from a 25% perfect DNA fragment, you’d have to screen 11 colonies.

You can use the table below as a guide for deciding how many colonies to screen for a given percent perfect.

If assembling multiple fragments, this effect is multiplicative, so when assembling two fragments of 50% perfect each, the combined percent perfect would be: 0.50 * 0.50 = 0.25, i.e., there is a 25% chance that any one colony picked contains the exact (combined) sequence. For a > 95% probability of finding the correct clone in this scenario, we recommend picking 11 colonies. Although this model is a useful guide for most cloning situations, it is obviously an oversimplification of biology; an important caveat is that if your sequence is toxic (or even modestly burdensome) to E. coli, fewer colonies might contain your perfect sequence than you’d expect based on the percent perfect estimate of the DNA Fragment(s) you’re attempting to clone.

Technical Tip!
Don’t just pick the big colonies. Sometimes the plasmid makes E.Coli grow slower. We recommend surveying a mixture of small and large colonies, if available.

What influences percent perfect?

Ansa DNA Fragments are manufactured by synthesizing one ultra-long oligonucleotide containing your sequence and then amplifying it to produce dsDNA. Errors can arise during oligo synthesis and during amplification.

The graph below shows the relationship between length and percent perfect for DNA fragments that originate from the oligo synthesis process (i.e. assuming no substantial burden of additional errors from amplification).

Note – chemical DNA oligo synthesis, in comparison, typically has 99.4–99.7% average stepwise yield for oligos up to a couple hundred bases. Ultra-long oligos are very difficult to produce using chemical synthesis due to DNA damage that occurs during the reaction cycle. Other gene synthesis vendors who use chemical DNA synthesis to produce gene fragments can enrich perfect molecules in a sample by using an error correction process that depletes heteroduplex molecules after a melt/reanneal step; however, complex sequences cannot be error corrected using these methods, so Ansa does not employ them.

How do we measure percent perfect?

We measure percent perfect using long-read sequencing, where we then align reads from a particular sample (filtered to have essentially no sequencing errors) to the expected sequence to estimate the fraction of molecules that contain the desired sequence.

Ansa’s minimum mass spec (yield) is 150 ng and average mass spec (yield) is 1.5 ug.

IDT is happy to help support questions around your order. Reach out to IDT customer service:
US/Canada: amr-custcare@idtdna.com
Europe: emea-custcare@idtdna.com
Asia Pacific: apac-custcare@idtdna.com

To locate the list of vectors, their descriptions, vector maps, and sequence files, please visit the Catalog Vector page.

Yes, Ansa has an early access program for custom vector onboarding. You can find more information and complete the custom vector form here.

The standard choices for sequences up to 7.5 kb are pANSA-Amp-v1.5 and pANSA-Kan-v1.5. Our most reliable vectors for long, complex, or toxic sequences up to 7.5 kb are pStable-Ind-Amp-v1.0 and pStable-Ind-Kan-v1.0. All sequences greater than 7.5 kb will automatically use the pStable-Ind-Chlor-v1.1 vector.

Ansa has removed chloramphenicol resistance from our list of standard vectors. It is available only for products 7501-50,000bp. If you are interested in continuing to use a chlor vector for standard products, please reach out to our customer support team.

Ansa no longer offers pAnsa3 (Kan) or pAnsa3 (Chlor). The standard choices for sequences up to 7.5 kb are pANSA-Amp-v1.5 and pANSA-Kan-v1.5. In order to compare the new vectors with the previous vectors, you can download the legacy vector maps and sequence files below:

• pAnsa3 (Kan): Vector Map | Sequence File
• pAnsa3 (Chlor): Vector Map | Sequence File

We provide 7,501bp – 50,000 bp in pStable-Ind-Chlor-v1.1, a single-copy vector that can be induced to mid-copy number using arabinose. Very large plasmids can be difficult to propagate in E. coli using higher copy vectors. This backbone ensures more reliable propagation during cell growth while enabling you to obtain a much higher yield than you could obtain with a single-copy vector. This higher yield is achieved by inducing the copy number after most of the cell growth has already happened.

We aim for competitive turnaround times (TAT) depending on your size and format.

• For fragments (200-900 bp), we ship in as few as 4 business days. 
• For clones (100 bp – 7.5kb), these are shipped in as little as 11 business days.
• For clones (7.5kb – 50 kb), these are shipped in as little as 25 business days.

Shipment times are dependent on when your order is placed. Reach out to your account manager or email us at customerservice@ansabio.com for more details.

Ansa can synthesize challenging sequences, including non-coding elements like promoters, ITRs and poly(A) tails. Our focus is on the synthesis of complex sequences that include features such as very high or low GC content, multiple repeats, and sequences with secondary structure. See our tech note for more details.

Ansa’s gene fragments are shipped with Forward and Reverse Adapters used to amplify the sequence and produce dsDNA. These Adapters can be removed based on each user’s needs by adding cut sites to the ends of the target sequence, or by using PCR amplification with sequence-specific primers.

Ansa takes Biosecurity seriously. We are a member of the International Gene Synthesis Consortium and adhere to their Harmonized Screening Protocol. All requested sequences are screened according to the NSTC Framework For Nucleic Acid Synthesis Screening before being synthesized. View Ansa’s Self-Attestation Letter.