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Molecular cloning

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I'm having trouble to clone some genes on DH5alpha E. coli strain. When I sequence the insert, I can only get 600 bp instead of the original size of the insert (1,2 kb). Does anyone know if this strain has restriction enzymes that can cut the insert?

Did you sequence the inset or just determined size? The strain is restriction deficient. Most likely the ligation did not work as expected, or you had a mix in fragments and the smaller gets preferentially cloned etc. etc.

  • 1 month later...

That means you got the insert but you cannot sequence it because it gave you just a 600bp piece? Then you may use another primer to continue the work, better to have some regions in overlap with the first 600bp, then you will get your first 600bp, and a second piece of another 600bp with some overlap so as to enable arrangement of fragments.

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