Dear Rick

I sugest doing subcloning first. I think it is better if you clone your blunt ended pcr product into a cloning vector which has been cut  by blunt-end-maker restriction enzyme like EcoRV. After succesfully cloning in this vector, you should digest your vector which contain's your insert by salI and speI. Purify your digested insert from gel and use it for cloning in your main vector that bas been cut by these two enzymes too. In this way you are sour that your insert has got bouth stichy ends that you need.Doing all these things may take a little more time but belive me it can guarantee your succeed in cloning. By  the way good luck.

Hi Rick, from what you said you follow exactly the protocols and all the step, except for the electroporation (I do not have an electroporator in the lab) and few other things I perfomed my cloning exactly as you did.

I thougth that the problems could be the digestion step, the ligation step or agar plate step. You have very good cells, no problems with transformation, you use TOPO cloning (good choice) so, in general, it is easy to digest insert without problems. You use a negative control, transforming your double digest vector, when you said "ligase control: no insert", I suppose!? So we are sure that you double digest very well your vector, you do not have false positive colonies from uncut vector.

My question/advise are:

Can you check problems with antibiotic? Only to be sure.

When you extract from gel and clean the plasmid (also the insert!!!) do you have some problems with the pH (I don't know if you use the Qiagen kits)? Maybe you can have some problems after the first wash and the second gel extraction (can you try to perform the double digestion at the same time or you have problems with the buffer?)

Do you use the restriction enzymes in other experiments?

Another thing is the ligase, you follow the protocols I supposed, maybe you can increase the time (I don't known what you used, in general I perfom ligation at 4°C ON). Can you use another ligation kit?

My other question is about the volume of ligation and the amout of volume you use in the trasformation, you use the high ratio of 1:5, how much ng of vector and plasmid you transform? It is important to not put a lot DNA or you can inhibit the reaction.

Maybe you have just check all this thing, I only try to find something to point out about the steps that you do not describe in your question.

Hope the best to your cloning

Thank you for the suggestions. Rick thanks for the question, I too have the same combination SalI/SpeI for cloning. My protocol is a PCR based cloning protocol, can I double digest my PCR product (which has restriction overhangs) with Sal1 HF (Neb) and SpeI HF (NEB) and keep it for digestion overnight. Since doing a sequential digest and purifying my PCR product after each digestion is going to cause a loss in the amount of DNA required for my subsequent ligation. I have also learnt SalI is a rogue enzyme and doesn't digest PCR products well.

My previous study using the combination both for insert and vector (double digest  both) did not yield results. The vector religated. I  had used a 1:1 combination of vector and insert .For vector a sequential digest followed by gel purifications and dephosphorylation of vector will be attempted.

Will this work out ?