Frequently Asked Questions
Can I order the PCR (Yellow 0.2 mL tubes) and circularization (Blue 0.2 mL tubes) buffers separately?
Yes, any of our individual reagents are available for purchase, please contact us support@FirstBiotech.com with the quantities you need.
What is the maximum mutation size that I can introduce into my plasmid using GenEdit™ kit?
The upper limit for insertions depends on the oligonucleotide synthesis and purity, but up to 120 adjacent nucleotides can be easily synthesized and inserted without HPLC or PAGE purification. The upper limit for deletions is around 10,000 to 12,000 continuous nucleotides.
Can I use primers that are not phosphorylated at their 5’ ends?
Yes you can, GenEdit does not require 5′ phosphorylation of primers.
Is it possible to use GenEdit™ to introduce mutations into BACs, YACs or PACs as vectors?
GenEdit™ works with any circular DNA as long as the mutation of interest is surrounded by unique restriction sites that are no more than 10,000 base pairs apart. The artificial chromosomes are generally too large to contain unique restriction sites; therefore the First Mutation technique cannot be applied to these systems.
My plasmid is 30 kbp in size; do I need to sequence my full plasmid after introducing my mutation of interest to check for unintended mutations?
No. The First Mutation technique does not rely on making a copy of the full plasmid in vitro. Therefore, it will not introduce any unwanted mutation(s) in the backbone of your plasmid. The DNA polymerase is used in First-Mutagenesis to copy only a small region containing the mutagenesis site, not the full plasmid. Therefore, only that region should be verified by sequencing.
What is the error rate of FBT HotStart High Fidelity DNA polymerase used in GenEdit kits?
It is 4.5×10-7. This applies only to the site of mutagenesis in your plasmid, not the full plasmid. GenEdit technique does not introduce any unwanted mutations in the rest of your plasmid (0% backbone error).
What is the best software to design my primers and select my restrictions enzymes?
First Biotech has developed a special software tool to handle GenEdit projects. It identifies the proper restriction enzymes and primers using an interactive interface. To use the tool click here.
We have an active mutagenesis program at our institution; can we get an institutional discount?
Yes, for volume discounts, please contact us at support@Firstbiotech.com.
I received the kit on dry ice. Is it OK to store the kit in a -20 ⁰C or -80 ⁰C freezer?
With the exception of the bacterial cells, all components should be stored at -20 ⁰C. The FBT5α Super Competent cells must be stored at -80 ⁰C to maintain viability.