The mutagenesis of CAST was performed using primer pairs (table S4). The target amino acid position was coded by NNK (sense strand), where N = A, G, C, or T and K = G or T. The whole-plasmid PCR method was performed using Proofast Super-Fidelity DNA polymerase with the plasmid DNA isolated from the second round of FACS enrichment as the template DNA using the following PCR thermocycling profile: 95°C for 3 min, 30 cycles of 95°C for 15 s, 55°C for 15 s, 72°C for 5 min, and 72°C for 10 min. The PCR products were digested with DpnI to remove the parent plasmid and then were cleaned using a DNA purification kit (Shanghai Life iLab Biotech, China). The resulting DNA mixture was transformed into E. coli JM107* cells for screening.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.