Construction of a genomic DNA library by monkey6

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									Construction of a genomic DNA library


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 ZAP 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
37C.




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 37ºC. 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 37ºC. 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 4ºC 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 l XLOLR cells and
incubated at 37C for 15 min. After addition of 300 l NZY broth, the mixture was
incubated at 37C for 45 min. Cell mixtures were plated onto LB plates containing
50 g/ml kanamycin and incubated overnight at 37C.          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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