Blunt End Cloning DNA

[pipette_man]

Hi everyone,

I am having blunt end cloning issues.


Does anyone know tips for this?

I have done the following.

PCRd my insert and filled the ends with Klenow. I also did this with my vector.

I phosphorylated them just in case and then ligated.
Any tips for efficient ligation? I think thats my problem

Thanks

[dnamolecule]

Hello there,

You are phosphorylating both insert and vector?

If this is the case you will be getting self-ligation products of insert-insert and vector-vector competing with your main ligation:
insert-vector.

Make sure you dephosphorylate your vector with SAP or Antarctic Phosphatase (NEB) works amazing.

Then just ligate your vector and insert with a super DNA ligase from NEB and you are set. Make sure you incubate long at 16C or 12C.

Cheers

[postdock]

Blunt end digestions can have several major optimization steps.

One is ensuring complete digestion of your vector.

Ensure complete digestion by allowing the digest reaction to go for a long time or add fresh enzyme + buffer every few hours until completely digested.


Another step is proper preparation of your insert.


Another is dephosphorylation of the digested vector.

I would suggest you use a phosphatase for DNA such as SAP (Shrimp Alkaline Phophatase) or antartic phosphatase.


SAP will remove the phosphates at the 5' end of the DNA strands on both ends of the digested vector which prevents it from self-ligating to its self through its 3' OH groups.

Some people use CIP Calf Intestinal Alkaline Phosphatase (or CIAP) but to be honest it can be a pain in the buns to inactivate. SAP can be inactivated in 15 minutes at 65oC and is compatible with many restriction buffers.

Ensure all DNA is prepared fresh and not old, or exposed long to UV.

Good luck!