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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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