Tuesday, February 16, 2010

hints about plasmid digestion



APE is a very useful tool for selection of required restriction enzyme.

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Protein contamination in the plasmid DNA solution doesn't supposed to effect the restriction reaction (though A260/280 is better to be higher than 1.8).

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TE Buffer is not recommended as an elution buffer for plasmid DNA that will be digested, although it provides a long time storage conditions by chelating the Mg+2 ions from the DNA (Mg+2 and other ions makes the DNA more prone to degradation). 

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After the digestion reaction completed,  oncentration of the loaded volume should be calculated and it shouldn't be more than 75 ng/ul. If there is very concentrated plasmid in the wells, linearized DNA will run slower than expected. 

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Molecular Biology Vector Sequence Database is a useful link for whole sequences of several vectors. 

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