Vector Cloning

At VectorBuilder, a combination of various molecular biology techniques is utilized to generate custom vectors based on customer needs. While expression vectors are typically constructed using Gateway cloning or Gibson assembly, shRNA and gRNA vectors are cloned using a ligation-based approach to provide you with your desired vectors at the lowest prices and fastest turnaround times utilizing our highly optimized high-throughput cloning platform. Additionally, we routinely use a variety of other methods including de novo gene synthesis and Golden Gate cloning as needed, on a project-by-project basis.

The origin of replication on your plasmid is for low-copy replication

The number of plasmid copies per bacterial cell is determined by the origin of replication on the plasmid. Some origins have inherent low copy number. Check the copy number of the origin of replication on your plasmid. For low-copy plasmids, increase the amount of E. coli culture for plasmid DNA prep in order to obtain satisfying DNA yield.

Check the copy number of the origin of replication of plasmids supplied by VectorBuilder

The volume of bacterial culture is too low for plasmid prep column

Please check the binding capacity of your plasmid prep column and whether your plasmid is high- or low-copy plasmid. For mini preps, we recommend that you harvest 1-5 ml of overnight bacterial culture. For maxi preps, if the plasmid is high-copy plasmid, we recommend using 100-150 ml of overnight bacterial culture; if the plasmid is low-copy plasmid, we recommend using 300-500 ml of overnight bacterial culture. Typically, for high-copy plasmid, ~5 ug of plasmid DNA can be extracted from every 1 ml of culture in mini prep and ~500 ug of plasmid DNA can be obtained from 150 ml of culture; for low-copy plasmid (e.g. pET), 1.5-2.5 ug of plasmid DNA can be harvested from every 1 ml of culture in mini prep and 150-200 ug of plasmid DNA can be obtained from 150 ml of culture.

Only a fraction of bacteria in the liquid culture contain plasmids

Some antibiotics, ampicillin in particular, degrade fast in liquid culture. As a result, bacteria that do not contain plasmids can propagate to a significant fraction of the culture, causing poor yield of the plasmid prep. To avoid this, please prepare ampicillin containing growth medium freshly before use and make sure that enough ampicillin is supplied. Also, when culturing ampicillin-resistant bacteria, do not let the liquid culture saturate for too long before harvesting. Besides insufficient antibiotics in the culture, extracting plasmid DNA from very old culture can also result in low yield, since many bacterial cells are dead and plasmid DNA they contain is degraded. Therefore, try to extract plasmid DNA from fresh culture. If plasmid prep cannot be performed immediately, you can spin down the bacteria and store the pellet in -80°C freezer for later plasmid prep.

The liquid culture is directly inoculated from E. coli stab culture

Direct inoculation of a liquid culture from the E. coli liquid stock or stab culture you have received from VectorBuilder can very occasionally result in low yield. We recommend streaking the stock onto an LB agar plate containing the appropriate antibiotic first, and then inoculating a liquid culture with a fresh colony growing from that plate. Detailed user instructions can be found by going to menu item Learning Center > Documentation > Stab Culture.

You have not carefully followed the manual of the plasmid prep kit

If you use a plasmid prep kit, please carefully read the manual before use. Improper operations can often lead to poor performance of the kit.

The mini/maxi prep column is low-quality

Some brands of plasmid DNA prep columns perform poorly or inconsistently for DNA preparation.

If our QC assays uncover a synonymous mutation, we will contact you for approval in moving forward with cloning, as this mutation will be unlikely to affect experimental outcome. However, if a non-synonymous mutation is found, we will discuss with you the best course of action for correction before cloning and downstream services are initiated.

De novo synthesis is required in less than 20% of our projects. Pricing for de novo synthesis typically correlates with the fragment length, sequence complexity (e.g., high GC content, simple repeats, or segmental repeats), and the number of DNA fragments to be synthesized. For specific pricing details, please submit a design request, and our team will provide a customized quote.