Problems with ligation and transformation - (Oct/31/2013 )

Hi there!

I have some problems with a plasmid construction, I´m using a pET vector (5 kb) where I have already fused my gen of interested and GFP. So, what I´m doing is first digest the plasmid to get rid of the GFP gen, then I do an agarose gel to check if it did cut and I purified the plasmid without the GFP. So then I double digest to insert this genes of aprox 500bp, I purified the digestion of the plasmids and the inserts (I obtained the inserts by PCR and purified them before the digestion) and then I continue doing the ligation, i left it at room temperature overnight. The next day I do the transformation in the competent cells. I do a negative control, but the problem is I´m having colonies in the negative control plate. I think positive and do some minipreps and a restriction to se if I have actually the insert, but no, the gel doesn´t show the insert... just a single scatterd among the line. I decided to run a gel of the minipreps (without digestion) and I´m having bands over the 10 kb band of the ruler, and in others preps It shows two bands aprox 10 kb and 5 kb each one.

So I don´t know from what are those bands and where is exactly my problem.

I need your help, thank you for your time :)

So you are removing GFP and the gene from the pET vector, and trying to ligate a different gene into the vector?

What restriction enzymes are you using?

What strain of competent cells?

I tend to leave my ligations at 4 degrees overnight. The overhangs will bind through hydrogen bonds (in the presence or absence of ligase) at temperatures below 80 (or thereabouts), but only the ligase is capable of creating the phosphodiester bonds. It's my understanding (rightly or wrongly) that ligase loses it's activity faster at higher temperatures, so if you're losing ligase activity before the overhangs of your molecules have a chance to anneal properly, your ligation efficiency will be reduced. Adding PEG to the ligation reaction can increase ligation efficiency.

Your negative control has colonies, thus your vector likely was not completely digested. Buy fresh enzyme, digest longer, treat with a phosphatase, etc. start with this, as nothing will work efficiently until you have the vector properly prepared. If you are confident that all was done correctly, skip the mini preps and do a full steam ahead approach. Do colony pcr on a lot of colonies, you will find a winner. With tough ligations, I have done colony pcr on as many as 40 colonies and only found 1 clone. Should it work this way? No! But it doesn't matter because I got the clone. Just something to consider...