problems with digesting insert from vector - (Sep/27/2005 )

Hi everyone,
I've been trying to digest my insert from a TA vector. It has an Nde1 and BamH1 site. i tried a double digest with NEB buffer for BamH1 and that failed so I tried doing a sequential digest using Nde1 first. But it refuses to digest . Bamh1 digests fine but as the vector has a BamH1 site I checked the presence of insert again using a colony PCR. My insert is definitely there. So why isn't Nde1 digesting ? Is there any one who has had a similar problem? I'd really be grateful for some suggestions, I'm pretty frustrated.
Thanks so much.

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hi
have you sequenced the ligation site to check that the RE site is there?
have you tried an enzyme from a different source? newer?

good luck
d

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THe NEB enzyme was the newer one, I'd tried with a different one earlier. i haven't tried sequencing the ligation site, I'll try that. Thank you so much.

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hi soraya,
i agree with dapo. you should change the enzyme. maybe you try another company. i have had similarly problems in the past with re from neb. maybe they are very sensitive or so but anyway, try another company.
good luck!

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hi

have u try digesting sequentially using BamHI, then NdeI?

or else, try digest each one (sequentially) with longer incubation time. or, use higher concentration of NdeI.

i think NEB enzymes works just fine.
good luck.

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