Ligation Failure - 20 colonies tested with no results. Need advice (Jun/23/2006 )

hello,

I am trying to express my protein using the pQE-30 vector. well recently, I have tried to ligate the gene into this vector using the following reaction mixtures:

1) insert gene: 4.4 microL
pQE : 1 microL
water: 12.6 microL
Ligase: 0.4microL
Ligation buffer: 1.6 microL
Total: 20microL

2) Insert gene: 14 microL
pQE: 2 microL
Ligase: 0.5 microL
Ligation buffer: 2 microL
Water; 2microL
Total: 20microL

After storing it at 16 deg for 3 hours and transforming DH5-alpha cells with the ligation mix, I initially don't get colonies. I tried transformation again and I got a lot of colonies (30+). However, when I did colony PCR to verify whether the pQE 30 contains my gene they had no bands. I tried like 46 samples and all gave me negative results. Even if there were, it was very faint. Surprisingly the ligation mix works well with my friend's genes which are of smaller size (100 to 500 bp) my gene size is 1700bp.... does it affect the success of ligation? It appears that concentration of ligation mix is specific to certain genes so how should I adjust the composition?

Would be really grateful if you could advice me on the ligation cos I am stuck here for a couple of weeks already. Thanks

Cloning is always problematic.

Do you perform a vector-only control to see how much ligation you have without insert?
Are you cutting with a single enzyme or two? If the answer is the former then do you use alkaline phosphatase?

Regrading the no colonies results - which competent bacteria do you use? We use heat-shock competent, and when the ratio of ligation mixture to bacteria mixture is too high (I use ~5%) then the transformation efficiancy drops.

Thanks for your reply.... we did not perform a control and I am thinking of doing one with the digested vector. Regarding my gene and vector they have gone through double digestion with BamHi and HindIII. I think for the transformation the I use 10% and it works for most of them. However, the concern right now is the ligation mixture cos the vector appears to be either undergoing self ligation and not my gene.

Thanks anyway really appreciate your advice.