I am currently trying to generate expression constructs of truncated proteins for x-ray crystallography. It is my first time doing cloning. I have had success so far in PCR amplifying my inserts using primers with NdeI, Bam HI, XhoI, and EagI restristion enzyme sites. My amplified inserts are running at the correct molecular weight as confirmed by agarose gel. I always ethanol precipitate my PCR produce overnight and resuspend the DNA pellets in 10mM Tris pH 8.0 and then do a double digest of the insert using NEB's restriction enzyme cloning tool. I typically digest my inserts for ~4-6 hours and then ethanol precipitate the digestion reaction. At the same time I do a double digest my pet28 or pet 15 vector with the same restricion enzymes under similar conditions, I have checked that each enzyme has linearized my plasmid on a gel before I add the other enzyme and then combine the digestion reactions. The uncut plasmid migrates slower than the digested plasmid and the digested plasmid runs at its expected molecular weight. I also ethanol precipitate my vector after the digestion. Finally, I use T4 DNA ligase from NEB and perform the ligation reaction. I then transform 200uL of competent cells with 10uL of the ligation reaction usually. I have tested my comp cells and used a control plasmid and was able to get good transformation efficiency with only 1ng of DNA and 50uL of comp cells but since the ligation reactions tend to give little colonies I have scaled up the transformation. I mini-prep some colonies from the transformation and have done double digests for over 100 colonies now and I do not ever see a band corresponding to my insert being there. Lately I have been doing a single digest to see if I can see the plasmid molecular weight increase as a result of the insert being there, but I do not think I see an insert also. For my most recent ligation I did a 7:1 molar ration of insert to vector in a 20uL reaction with ~20ng of vector. I have also tried 50ng of vector and 20:1 10:1, 1.2:1 ratios and still i get no insert. My most recent transformation for example gave 3 colonies for my insert reaction, 0 colonies for my control of no insert +ligase, and 5 colonies for my control reaction with no insert or ligase. I haven't digested the mini-preps yet but I feel like I will not see an insert again. I would say these results have been typical for my ligations, sometimes I get ~7-27 colonies for my reaction with the insert and 0 colonies for both of my no insert controls. When I test the colonies though, I do not see an insert or shift in molecular weight indicating the insert is not in my vector. Does anyone have any suggestions how I can get a clone successfully ?