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Very high insert to vector ratio during ligation.

Very high insert to vector ratio during ligation. - Molecular Cloning Forum

Very high insert to vector ratio during ligation. - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 03-25-2009, 07:38 PM
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Default Very high insert to vector ratio during ligation.



A technician recently made a mistake by adding way to much insert during a ligation-transformation procedure. The ratio was nearly 150:1 insert to vector instead of customary 4:1 ratio. The plate resulting plate did not produce any viable colonies. Members of the lab found this quite interesting and prepared several dilutions ranging from 1:1 to 100:1 and found that indeed when too much insert to vector or too little insert to vector produced fewer colonies with a normal amount of colonies appearing between 3:1 and 40:1.

We came up with a few ideas as to why this may be the case biologically.

Does anyone know anything about the cause of this or were we can look?

thanks
cw
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