NdeI: cutting problems?? anyone else?? seems silly! (fwd)

logan david.logan at plant-sciences.oxford.ac.uk
Tue Mar 12 11:22:07 EST 1996


>greetings 'netters!!
>Has anyone else experienced problems with NdeI (ie. 
>incomplete/non-cutting)?
>We have 2 lots from 2 different companies (BM and Gibco) and neither of 
>them are working. Trobleshooting (ie. altering buffer conditions--there 
>doesn't seem to be a consensus in the literature) has turned up nothing. 
>It is a mystery to us and the companies that supplied them. Even on 
>control DNA!!
>what is going on? Is there an NdeI god out there who has cast a spell? 
>All other enzymes are working perfectly.
>If you can shed any light on this problem, please let me know.
>Thanks in advance!
>--Heidi



>A colleague of mine told me that NdeI has an extremely short half life 
>about quarter of an hour)and she therefore adds extra units during the   
>course of the reaction.
>I've had problems using NdeI for cutting plasmids for subcloning i.e. 
>I've never been able to obtain complete vector digestion  and consequently 
>have to use CIAP even when cutting a polylinker with NdeI and another 
>enzyme to generate incompatible termini. Usually the second enzyme 
>works far better than Nde resulting in a high background of self ligated 
>plasmid.
>Hope this tip helps.

>Neil Harris,
>Plant Science Laboratory,
>St. Andrews University.

I concur with the above. In my last project I was using NdeI cut vector for 
expression cloning (ATG site) and the person who prepared the vector said 
the purity of the plasmid was vital. In the end she prepared her vector by 
cesium chloride gradients. She also added NdeI during the course of the 
reaction. However, when I prepared my PCR product by gene-clean of agarose 
purified band and cut it with NdeI (inserted restriction site in primer) it 
cut fine so....? It ain't so simple!?



David C. Logan																

*********************************
*	david.logan at plants.ox.ac.uk			*
*																															*
*	Department of Plant Sciences		*
*	University of Oxford										*
*	South Parks Road 													*
*	Oxford																								*
*	OX1 3RB																							*
*																															*
*	Tel.(direct): (01865) 275024		*
*																															*
*********************************




More information about the Methods mailing list