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restriction enzyme digestion problem - (Aug/22/2005 )

I have the correct PCR product at 500 b.
After enzyme digestion (NdeI & BamHI), the band smears up on the gel, and non-targeted large bands (6kb, 1.6kb) appears on the gel.

Anyone can suggest a way to avoid it?


i've used enz successfully in the past for cloning, did u gel purify it, what's teh amt of DNA u r using, use double amt of NdeI, this might help.
good luck


What are you trying to do? If you want to clone of your fragment I would advise cloning into something like topo first then cutting out the insert with your two RE. If you are inexperienced this will prove much simpler and faster in the long run.


PCR sequencing DNA

-Daniel Tillett-

Good idea. Thanks!


How are you checking you have the 500bp product? Might be a silly suggestion - but I have seen people attempting to guess on a gel - seems odd to be able to get a band of higher bp after you supposedly cut.... hence maybe your 500bp band is not 500bp?
smearing after resticiton enzyme suggests that you have genomic DNA or a massive amount of non homogenous DNA (ie not a PCR product)

The topo equip is good stuff - highly recommended.

edwina x


Hi, sorry, what is Topo? Is it TA cloning?


QUOTE (huotsing @ Aug 24 2005, 04:50 PM)
Hi, sorry, what is Topo? Is it TA cloning?

topo is a TA cloning kit yes. Its rewally user friendly and easy to understand.
Just put TOPO into a search and it should come up with it. - not sure if we are allowed to say the company name on these messages... but its an invitrogen


Thank you very much!!!