Construction and screening of genomic library from Raji cells

Objective To construct the genomic library of Raji cells and screen it by EBV DNA probe.Methods High molecular weight genomic DNA of Raji cells was digested by restriction enzyme BamHI.DNA fragments ranging from 9 to 23 kb were recovered by agarose gel electrophoresis,which were ligated with Lambda DASH II vector BamHI arms pre-treated with calf intestine alkaline phosphatase(CIAP).Ligated DNA was packed in vitro using Gigapack Ⅲ gold packaging extract.The library was plated on XL1-blue MRA(P2)host strain.Titering and screening of the Raji genomic library were performed.Results The primary titer of the Raji genomic library was 1.8×105 pfu/mL,while that of the amplified library was 2.8×108 pfu/mL.Plaques(1×105)were screened with 32P-labeled EBV DNA probe(EBV genome 5-3271),4 positive clones were obtained,and 1 of the 4 positive clones was picked out randomly for the second round of plaque screening.All the phage plaques were positive.DNA of the positive clone was extracted and was digested with BamHI.The length of the inserted fragment was 8.5 kb.Sequencing and BLAST analysis revealed that the inserted fragments consisted of the BamHI-W fragment at one end and clone RP11-665A22 on chromosome 15 at the other end.Conclusion The successfully established genomic library of Raji cells will provide a basis for cloning the sequences of the EBV junction sites and interpreting the mechanism of oncogenesis of EBV integration.