Construction of a genomic DNA library

Thursday, December 04, 2003

Description
Construction of a genomic DNA library

Procedure
Library construction

Two micrograms of genetically modified genomic tobacco plant DNA was digested with 60 units of the restriction enzyme, BamHI and ligated into the BamHI site of a pre-digested lZAP Express vector, which is part of the ZAP Express Predigested Vector Kit (Stratagene, USA). Packaging extracts were used to package the recombinant lambda phage following the instruction of the manufacturer (Gigapack III Gold Packaging Extract; Stratagene, USA). Of the resulting library, 3.0 x 105 plaque forming units (pfu) were plated onto NZY agar plates containing 5 g/l NaCl, 2 g/l MgSO4x7H2O, 5 g/l yeast extract, 10 g/l casein hydrolysate and 15 g/l agar (pH 7.5), using XL1-Blue MRF bacteria strain as a phage host and incubated overnight at 37C.

Library amplification

The library was amplified to prepare a large, stable quantity of a high-titer stock of the library. Aliquots of the library suspension containing 5 x 104 pfu of bacteriophage were plated out on 150 mm NZY agar plates and incubated overnight at 37C. The plates were overlaid overnight with SM buffer consisting of 5.8 g/l NaCl, 2g/l MgSO4x7H2O, 1 M Tris-HCl (pH 7.5) and 2% gelatine to allow the phage to diffuse into the SM buffer. The bacteriophage suspension from each plate was then pooled into a sterile container and cell debris was removed by centrifugation for 10 min at 500 x g. The supernatant was recovered and transferred to a sterile polypropylene tube.

Plaque lifting

The library was plated out at 50 000 pfu/plate on large 150 mm NZY agar plates and incubated overnight at 37C. A nitrocellulose membrane (Stratagene, USA) was placed onto each NZY agar plate for 2 minutes to transfer the phage particles to the membrane. The plates were chilled at 4C for 1 h before placement of membranes onto the agar to prevent the agar from sticking to the nitrocellulose membrane. A needle was used to prick through the membrane and agar for orientation. The membrane was denatured in a solution of 1.5 M NaCl and 0.5 M NaOH for 2 min, which was followed by neutralization for 5 min in 1.5 M NaCl and 0.5 M Tris-HCl, pH 8. The membrane was rinsed for 30 sec in a solution containing 0.2 M Tris-HCl (pH 7.5) and 2 x SSC solution buffer. The DNA was finally cross-linked to the membrane using an UV transilluminator.

Library screening

The genomic DNA library was screened by Southern blot analysis. Three DNA probes constructed from respective DNA subtraction products were labelled with a Gene Images random prime labelling kit (Amersham Life Science, UK) and used for screening. Any positive clones were picked and excised from the ZAP express vector as a recombinant pBK-CMV phagemid plasmid (Stratagene, USA). In vivo excision of the pBK-CMV phagemid vector was provided with the help of the ExAssist helper phage, which contains an amber mutation to prevent replication of the phage genome in the non-suppressing E. coli strain, XLOLR, supplied with the kit. Dilutions of the excised pBK-CMV phagemid vector were mixed with 200 ml XLOLR cells and incubated at 37C for 15 min. After addition of 300 ml NZY broth, the mixture was incubated at 37C for 45 min. Cell mixtures were plated onto LB plates containing 50 mg/ml kanamycin and incubated overnight at 37C. Plasmids of individual colonies were confirmed to contain inserts by digestion of plasmid DNA with the restriction enzyme BamHI restriction and detection of DNA fragments by gel electrophoresis on a 1% agarose gel in TAE buffer.

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Construction of a genomic DNA library