I want to detect a digested product of 62 bp in an agarose gel.
I am doing a Site Directed Mutagenesis and want to screen my colonies. The mutation introduced a restriction site XhoI. Another XhoI is already present 62bp downstream and therefore only way to detect my positive colony is to do digestion. Can anyone suggest a solution to this problem based on experience?
small sized dna are best detected on 12% - 15% polyacrilamide gel, low concentration dna can be detected with silver staining, if there is no possibility of PAGE 2 or 3% agarose can be used, the band may appear diffused and size estimation may be difficult
It would be better if you added more information like whether it is a oligo, pcr fragment or digested product
another alternative could be to design a primer ending at the newly created xho1 site and a reverse primer 200 bases away then run a pcr under stringent annealing conditions so that only the mutated sequence amplifies then you would have a larger fragment as a positive result but I like Richard's approach as well
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