CIAP absolutely required? - (Aug/12/2004 )

Hi guys,

stumbled upon this fantastic forum for molecular biology.

Would like to pose a question

I am currently trying to clone a fragment of DNA into the pCDNA3.1+.
Thing is I am using the same restriction enzyme on both sides of the fragment and hence corresponds to only one RE on the vector itself.

I was thinking whether the addition of CIAP is required, or should I just digest the vector and insert, gel extract the DNA and directly ligate them without the CIAP. Would that result in a lot of self-ligation? Also, will the ligation reaction work just as well?

Anyway, I find my CIAP not very effective in prevention of self-ligation. Have personally tried using it, after digesting vector, CIAP was added directly in the digestion mix after an overnight digest and incubated for 30 minutes to 1 hour. After ligation and transformation, clones collected were PCRed screen but mostly showed nothing except the vector's uncloned size fragment. Was contemplating shrimp AP instead for next purchase.

Would appreciate any input on this matter.

Thanks and Best Regards,
tltan

-tltan-

QUOTE

CIAP absolutely required?

Hi tltan,

I would say "yes"

If you use just one RE, clones with an insert are typically rare compared to religated vector. On the other hand, CIP doesn't solve this problem completely, so where appropriate a double digest would be preferred.

There could be some things that cause trouble in your case:

1) If you have a 5'-overhang, 1 hour at 37 °C is ok. If you have a 3'-overhang, try to use 30 min at 37 °C and 30 min at 56 °C instead.

2) In any case inactivate CIP by incubation at 85 °C for 15 min and use a kit to purify your vector, otherwise CIP will continue its task and dephosphorylate your insert, too.

Greetings
CKM

-CKM-

hi!

CMK is right!

I'd say YES, too.

But maybe you are willing to change CIP for Shrimp Alkaline Phosphatase - works much better in my hands!

mike

-jadefalcon-

Hi CKM and jadefalcon,

Thanks for the reply. Will definitely try the technique recommended.

In our lab, we never inactivate CIP and just PCR purify using Qiagen PCR purification kit.

Also, didnt know that the timings of CIAP treatment is so critical.

Cheers,
tltan

-tltan-