Swapping promoter of destination vector not working. Please help! - (Oct/07/2014 )

Hello, 

I am new to restriction enzyme cloning and am having trouble with what should be something simple. I am trying to make a complementation line driven by the native promoter. I have done the ligation with varying ratios of vector to insert (3:1, 5:1, 7:1, 10:1, 15:1, 20:1)- all with no success. At first, I was getting no colonies after transforming. I have to use ccdb resistant E.coli because I am trying to propagate a vector (Gateway destination vector). I figured that this was due to the poor efficiency of our old competent cells and ordered new ones from Invitrogen. I did the transformation and I GOT COLONIES! However, when it came to the plasmid prep, diagnostic restriction digest and sequencing, I found that none of the colonies I chose were positive. 

I am using pMDC123 (~10,200 bp). I removed the portion of the vector I wished to replace by performing a restriction digest with AscI and HindIII. I did this at RT overnight, followed by inactivation of the enzyme at 65 degrees C for 10 min.  I ran it on a gel and gel extracted the fragment that I wanted. The insert I am trying to use I amplified from gDNA (it is ~ 1.5 kb) and ran on a gel. I gel extracted the fragment and did a restriction digest to create sticky ends for cloning . I was sure when I designed the primers that I had the RE sites in the right orientation in respect to the vector. I was also sure to not use a RE that will nick my fragment where it is unwanted.  I digested the fragment the same way, RT overnight with inactivation at 65 degrees for 10 min. I performed the transformation and got incorrect clones. 

I feel like I have tried everything. Ordered new ligase, tried different brands of ligase. Remade vector and insert, tried varying lengths and temperatures for ligation reaction, varying concentrations of vector to insert...I am at a loss now. I have been trying to get this to work for 2 months with no success! Please help....:( I am at a complete loss and cannot move on from this. 

P.S. I am using NEB T4 ligase. 

Thank you so much for your time!

It's not really possible to tell without a lot more information about exactly what you are doing. Some quick possibilities include DNA damage from UV exposure during gel cutting, and incorrect design of the primers for PCR (lack of the extra 5' bases in front of the RE site).

I don't know why you are doing the restrictions at RT -- both enzymes work optimally at 37. And inactivation takes 80 for 20 minutes.

The problems are rarely with the ligation, and much more frequently witht the DNA preparation before that.

You could test your PCR fragment digestion (which is difficult to test other ways) by cutting with each enzyme separately, then ligating the fragment to itself. You should get a double length fragment when the two fragments ligate to each other.

Make sure you are purifying your PCR product before cutting with your restriction enzymes.

How many extra bases do you have at the ends of your primers 5' of the restriction sites you are using?  If you have the HindIII sequence as the very first or last bps of your amplified fragment it may not cut (according to the NEB site).