I have been trying to construct a new plasmid to be used in cloning of a restriction modification gene from B.mega to ER 2925. The new plasmid is a construct of a common 3kbp plasmid and a 4.3kbp fragment of Lambda phage. The plasmid was linearize with a blunt cutter and dephosphorilated, the lambda was cut with two 5' overhanging restriction enzymes and run on a .5% agarose gel. The fragment was harvested out of the agarose by mean of B-agarase and and the overhangs were filled in with a quick blunting enzyme from New England Biolabs. The two pieces of DNA were then ligated with a standard over night ligation and transformed into ER 2925. The transformants were plated on the correct antibiotic selective plates. There were colonies that grew on antibiotic selective plates and all controls were as to be expected. The problem is when the new plasmid is harvested from the ER 2925 and run on a gel, the native form only gives rise to one band at around 10kbp instead of the normal three bands. When the DNA is cut with a common enzyme (HindIII or BamHI) the gel gives rise to a smear. I can use the DNA that was recovered from the plasmid prep to transform more ER 2925 but it gives the same result. I have done this ligation three times and the same results occur. I know with a blunt end ligation I might get two pieces of the original plasmid and one insert, that would explain the size, but I do not understand why it would become unstable when cut or why I am only getting a single band when run native on a gel. Any help or ideas would be welcomed. Thank you
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