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I have designed the primers for PCR cloning, but i was not getting any colonies after the ligation. One of our colleagues said, that the restriction digestion might not be working as there is no linker sequence before/after the restriction site in the primer. What measures should I take to be successful in the cloning?

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  • $\begingroup$ You can add the restriction recognition site directly next to your sequence, this is not a problem. However, a lot of restriction enzymes have a lower or no activity, if the restriction site is directly at the end of a DNA strand and require some additional bases here. $\endgroup$ – Chris Aug 23 '16 at 6:52
  • $\begingroup$ i was using bamhI and hindIII for digestion ... do i need to order a new primer set ... and is there any specific linker sequence for respective restriction enzymes? $\endgroup$ – saikrishna s b Aug 23 '16 at 7:42
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This is the website you need, and yes, most of the time it's better to have several extra bases at the start of your primer.

https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

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