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Re: [ccp4bb]: Cloning !!!



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Hi,

 that sounds all great. Just wanted to add another observation I had
with NdeI. The number of required overhang bp's mentioned in the NEB
catalogue was in my hands too short (got exactly nothing for the
ligation). I only had success after I used 12 bp overhang for NdeI.

	Good luck,

		Manuel

Gregory Verdon schrieb:
> 
> Hoi,
> 
> For the transformation, you should have a positive control ! Try to transform
> 
> cells with the original pET-29 vector. Then, you should also check your
> 
> enzymes. Again, you can do a control for each enzyme on the original vector
> 
> (separated experiments). Put both experiments on gel against markers and
> 
> the undigested vector. For each enzyme, you should get one linear fragment.
> 
> For the PCR product (i guess you get it from a PCR experiment), it is more
> 
> difficult to check. My guess is that you have a problem with the NdeI enzyme
> 
> If i recall it correctly, NdeI needs quite quite a lot of bases upstream of the site
> 
> to be able to cut (let say it should be more efficient). You should check the catalogue of
> 
> the company from which you bought the
> 
> enzyme. You should also check your disgested fragment on gel to see if you don't
> 
> get some STAR activity by the enzymes.
> 
> For theligation experiment, be sure that you have more insert than vector and try the ligation
> 
> at room temperature and in the cold room overnight. Again you can check the ligase on
> 
>  with the vector cut with only one enzyme and then do the transformation. The last possibility
> 
> i see could be that you made a mistake for the primers (if PCR) !!!
> 
> Good Luck,
> 
>                             Greg
> 
>   ________________________________________________________________
> 
>   Gregory Verdon
>   Laboratory of Biophysical Chemistry
>   Department of Chemistry
>   University of Groningen
>   Nijenborgh 4                        tel   : +31(50)3634384
>   9747 AG Groningen                   fax   : +31(50)3634800
>   The Netherlands                     e-mail: g.verdon@chem.rug.nl
> 
>   http://www.xray.chem.rug.nl/
>   ________________________________________________________________
> 
> 

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