Gel for my ligation - (Aug/22/2014 )

 Hi all,

I can not get colonies at all time. So I run my ligation product. Please help me to fix my problem. COLONY come on!

Also, I have some question.

1. I design my primer with two REs site, and I design extra 3 bases at the end. I am not sure the 3 bases were enough to Xho1 and EcoR1. NEB website shows 50% efficiency with three bases.

2. Because the two REs was so close in my vector, i.e., Xho1 (978), EcoR1 (931). So I just wonder if I need to do separately digestion step to prevent from the two competitive REs reactions. And I have done that, the attachment showed the result.

3. I also do one step, digest the pcr product with one enzyme, then put the ligase, if both enzyme and ligase were worked, pcr bands should be doubled. According to the gel, I found my PCR product with EcoR1 digestion was slowed migration then pcr product with Xho1. I think the band should locate in the same position. Do I need change the new REs?

4. Do I need stop now? Redo it? Or I can do next transformation?

Ps: Gel result has been attached. Lane 2 and lane 3 were not so clear because I just use 20 ng vector for 20 uL ligation system, then I just load the 5 uL on the gel.

From your gels it looks like the plasmid has been cut efficiently, however you didn't show digests that only contained one of the restriction enzymes (RE) - so there is no way of telling if one of the REs is not cutting. There is no way to tell if your PCR product is cutting efficiently, which is quite likely the problem you are experiencing. Ligation of the inserts to themselves won't work efficiently in RE buffers as they don't contain ATP usually, and they are often the wrong ionic strength.

You could easily re-order your primers, just add 3 more bases on the 5' end to ensure that there are enough bases for efficient cutting, which would make the cut more certain and hence the ligation more likely. Running a digest with REs still bound to the DNA could account for the slightly slower migration of your "ligated" insert. If it had properly ligated you would expect that it would run double size.

Note that if you are doing your ligations properly (small amounts of DNA work best, 20 ng backbone is usually considered the upper limit), then you are unlikely to be able to see if on a gel after ligation.