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Nilanjan Bhowmick AIR 3, CSIR NET (Earth Science)
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You perform a restriction of a pgem-t plasmid vector with ecor1 to remove a cloned fragment. you predict that only 2 fragments will be generated from this diges
You perform a restriction of a pGEM-T plasmid vector with EcoR1 to remove a cloned fragment. You predict that only 2 fragments will be generated from this digestion since 1) the EcoR1 sites in the plasmid flank the cloning site and 2) no EcoR1 sites exist in the cloned fragment. However, agarose gel electrophoresis revealed that 2 additional bands of unexpected sizes were also generated. You discover that the restriction buffer that you used in your assay is not recommended for EcoR1. Given this information, name 2 factors that would generate your unexpected results and name and explain the exact mechanism responsible for these data.
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Krishan k jakhad
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this may be due to star activity that may generate off target products. although reaction conditions including temperature and buffers are very important for high efficiency of restriction endonucleases.
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Krishan k jakhad
also since EcoR1 recognised G/AATTC it occurs on average 4^6 bases..repeat 4 kb around
could you please elaborate on this point..