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how do you check your restriction enzyme is working? - (Oct/12/2005 )

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do you mean after second digestion of my PCR, no need to purify it and directly use it for ligation?


No you should gel purify it after your second digestion. I still recommend that you put the PCR product in a TopoTA vector first like other people have suggested on this board.


Sequential digestion is helpful if the restriction sites are very close by. I had two consecutive sites with only 1 base in between. I digested with first enzyme (which was less efficient) for 1 hr in the common buffer, then added the second enzyme as well as little same buffer and continued incubation for two hrs. Still as a caution I did dephosphorylation with Roche Alkaline Phosphatase. and I got successul ligation with zero colonies on control plate!


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