Producing a P1 bacteriophage library from pine: isolation and cloning of very high molecular weight DNA.
We have generated a genomic P1 bacteriophage library using Monterey pine (Pinus radiata) DNA. We first developed a method for isolating from pine tissue the very high molecular weight DNA necessary for the preparation of libraries requiring large inserts. The method involves protoplasting the cells, isolating nuclei and lysis in a high concentration of detergent. Fragments of greater than two megabases in size are produced in solution. Modifications introduced to the protocol for library preparation and for P1 plasmid isolation are described.
Gorman
S W
SW
Case Western Reserve University.
Roberts-Oehlschlager
S L
SL
Cullis
C A
CA
Teasdale
R D
RD
D001435:Bacteriophages / Q000235:genetics*; D002467:Cell Nucleus; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016521:Electrophoresis, Gel, Pulsed-Field; D015723:Gene Library; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010944:Plants / Q000235:genetics*; D010957:Plasmids; D011523:Protoplasts
D004247:DNA