arunee

Hi,

I come up with failure here

I did the same above protocol with the vector plus shrimp alkaline phosphatase, then purified.
the insert was amplified using proof reading taw with primers designed with BamHI and XhoI at both ends.
PCR product was purified and digested with BamHI and XhoI. Then purified again before ligation.

Ligation and transformation was performed according to the above post.

For the transformation result,

1. positive control (uncut vector): numerous colonies
2. negative control: no colony
3. ligation plate: 6 colonies with satellite colonies
4. cut vector: 6 colonies with satellite colonies
5. Insert only: no colony

Usually when I have a few colonies with satellites, I always get only empty vectors. So it seems like I have undigested vector on my plate here, I guess.
I can see that the digestion was incomplete but I was wondering how is that possible that no a single insert could ligate to some digested vectors.
Maybe something wrong with the ligation? I have just bought a new tube of T4 ligase last year and we used it not often until a few months ago that I tried a hundred times of cloning.

Please, help

pDNA,

Thank you for your suggestion. I am going to start the whole thing again and will do those control you suggested.
I will keep you posted.

If you have any other suggestions, please kindly let me know.

pDNA,

I tried cutting my vector with BamHI and SacI, and XhoI and SacI. These pairs of enzyme produced ~400 bp fragment which I could see clearly on the gel. So I think the plasmid is good with both BamHI and XhoI.

BTW, while I am waiting for my sequencing result, I have just tried cutting out my positive clones with other enzymes yesterday. It turned out that I don't have any insert in there but an empty vector!

Please help,

Jason_LuYZ,

yes I have already sent those positive clones for sequencing and it takes like 2 weeks to get the result! I just can't wait for it.

I used to do PCR on the bacteria to get the insert using proof reading taq, and the primers with the restriction sites at both ends (BamHI and XhoI). the cloning was successful. But I was just worried that some bases could go wrong along the sequence so my PI suggested me to commercially order the insert which is synthesized in the pUC57 plasmid and did the above protocol.

I am still wondering why there was no positive band from PCR on pUC57 plasmid while I can cut my insert out of it and the sequencing result showed the exact sequence I expected. Is there anything that can go wrong with the pUC57 plasmid I have?

Thank you so much for your advice.