NdeI double digestion - (Jun/07/2006 )

Dear All,
I have a 200 b.p. PCR product with an NdeI restriction site at the 5' and 3' ends plus 6 additional arbitrary bases at the termini. When I double digest with NdeI it does not work. In fact when I run the treated sample on a gel next to untreated, the treated sample travels a slightly shorter distance which would suggest the NdeI is still bound to the insert (like band shift assay).
In the past I have got this double NdeI digest to work and cloned into a vector singly digested at its NdeI site. Of course the insert could ligate in both orientations so extensive colony PCR screening was required to find the correct orientation. But now I want to do the same thing with a different vector and cannot get it to work. What I have also tried is digesting the mentioned plasmid I succesfully prepared as this would have the two NdeI sites. Rather than seeing the 200 b.p. insert band on a gel I see the vector band slightly shifted suggesting it is only singly digested and linearised.
This is very frustrating because I have got this to work before.
Anyone have any ideas please?
Chichibabin

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Have you tried cloning the NdeI fragment you just cut? Your cut product should migrate faster on a gel, however, you would have a very hard time seeing the migration of 12 base pairs of a linear fragment.

I suggest testing your cut, by cloning. If you are cloning a single digest fragment into a single digest vector, you will most certainly get self-ligation and no recovery of a fragment. Try a greater mol ratio of insert to vector (5:1) at least.

As far as orientation of insert - save time and use different restriction sites...

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Hi,
I have tried cloning the insert and get zero colonies. Also by treating the singly digested vector with phosphatase I get zero background on the vector control plate so vector self ligation is not a problem.
I have to use a double NdeI digestion to obtain the construct I require.
Sat

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hi,
since u hav done phosphatse,try gel eluting it........ie.try to gel elute the cut band....and run a little along side the uncut band as control n c if it helps.....
n then gel elute ur fragment....
now for the ligation part....try overnite at 16c or at lower temp overnite............and yes while doing transformation take all of ur reaction mix,now this time scale up ur reaction mix and use all of them at one go......
i had that problem and this worked fine....may b try changing your ligase and competent cell...this may help too...........
ok best of luck.....
nikz

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