Genetic manipulation of the BAC-cloned KSHV genome

To generate a series of KSHV helicase mutant KSHV genomes, mutagenesis of BAC16-RGB (RGB) was performed using a recombinant system as previously described. Briefly, a linear DNA fragment was amplified via PCR using the pEPKan-S plasmid as a template, which contained a kanamycin cassette, an I-SceI restriction enzyme site, and duplicated flanking sequences derived from KSHV genomic DNA (approximately 40-bp copy). The PCR product was treated with Dpn I to remove the plasmid template, and the purified PCR fragments were electroporated into the RGB-containing GS1783 strain, which had been induced at 42°C for 15 min. The recombinant clones were selected at 32°C on LB plates containing 12.5 μg of chloramphenicol and kanamycin (50 μg/ml). Positives clones were treated with 1% l-arabinose, induced at 42°C, and plated on LB plates containing 1% l-arabinose for secondary recombination. The resultant BAC was further confirmed through DNA sequencing.

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.