Cloning trouble - (Jan/15/2008 )
Hi everybody! This is my first time and I hope you will help me, please!!!
I have been trying to ligate an insert (1Kb) amplified by PCR in a plasmid (14Kb, pNL4.3), and all my results have been negative until now. Both, plasmid and insert, are previously cut with ApaI and AgeI. I am really desperate because I have tried many different conditions, always with the same result: nothing! 
I know that cloning sometimes depends on luck 
, but I hope someone could help me
Thanks a lot.
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1)give the ratio of insert/vector you are trying and also the final dna con of your ligation reaction.
2) also give the primers you choose, highligting the respective restriction site and no of extra bases at the 5' end of each primer.
3) and any other conditions
1)give the ratio of insert/vector you are trying and also the final dna con of your ligation reaction.
2) also give the primers you choose, highligting the respective restriction site and no of extra bases at the 5' end of each primer.
3) and any other conditions
2) also give the primers you choose, highligting the respective restriction site and no of extra bases at the 5' end of each primer.
3) and any other conditions
Hi Nagu!!! Thanks for your reply.
These days ago, I was trying the ligation reaction in a ratio of 3/1 (insert/vector), though I am thinking that maybe I should increase the concentration of the vector because of its great size, and beacuse I have tried differents insert of the same size. The last reaction has been done in a volume of 10ul, adding 4ul of insert and 4.5ul of vector (1ul of buffer and 0.5ul of T4).
Another thing could be that I am using the ligase`s buffer diluted 1:10, because if I use it 1x I will have troubles in the next step, transformation by electroporation. If I increase the concentration of buffer, I am increasing the concentration of salts and then the electroporation won´t be successful.
I hace read your answer for Mugu, I am right thinking on it! I hope you coul help a little more!
I use ligation buffer at 1x and don't have any problems with transformation by electroporation, I just dialyse the ligation product before electroporation (incidentally I forgot to do this last bit once or twice and I still had successful transformations). You haven't really got much to lose by giving it a shot, so that would be the first thing that I would try....
might it not be possible to use a set of less exotic restriction enzyme. AgeI in particular seems a little iffy from what NEB has to say about it.
Hmmm...
How many basepairs have you added around your restriction site on your primers? Restriction enzymes require their cut sites to be skirted by a minimum number of bp to cut effiently. Most often this is around 6bp but some enyzme require a minimum of 8bp.
How much DNA are you ligating? Can you give a number?
Do you gel purify your DNA? It is always a good thing to gel purify your DNA. Cuts out all the junk.
However be careful with the UV exposure. Your DNA should at most be exposed to UV for seconds. Minutes is too long. So work fast and work quickly. Also note what kind of transluminator you are using. Some illuminator have a short and long wavelenght setting. Short is bad as the UV light is more energetic and can cause crosslinking faster.
Are you certain your ligase is still working? T4 ligase and ligase buffer go off fairly easily. Take some of your ligated DNA and run it on a gel (use a narrow comb to help visualise the limited DNA you have). If ligation is working, and you have not lost your DNA, you should see high molecular weight bands denoting ligated DNA.
Thaks a lot Lauralee and Perneseblue!!!
I am going to try with buffer 1x as you suggested me lauralee, though I really think that the problem is at the ligation step.
The plasmid I am using do not need the addition of RE sites, it have them for its own. The same happens with the RE sites of the insert, so I really don´t think that troubles are here. I have tried many conditions, always increasing or decreasing the concentration of the vector, between 35 ngr and 245 ngr. It is true that the concentration of the insert has not been so controlled. I would like you to tell me the best realtion between vector and insert of these sizes.
After the action of the REs, I always purified the DNA either from a band of an agarose gel (vector) or from RE reaction (insert), using a kit for PCR product purification. For the other suggestions I can tell you Perneseblue that I take a lot of care of the exposure to UV nada the T4 is still working. I have some proofs of it.
Thanks a lot again!!!
14 ng of a 14 kb vector = 1.538 fmoles or 9.3 e 8 molecules.
1 ng of a 1 kb fragment = 1.538 fmoles or 9.3 e 8 molecules.
You need equal numbers of molecules or ends, so in this case the insert:vector ratio is 1 ng:14 ng but actually 1:1 for number of ends.
When you did a 3:1 insert:vector based on ng, you had 2.8 e 9 molecules of insert:6.6 e 7 molecules of vector. That's actually a 42:1 ratio of ends, not 3:1. That's going to favor concatamers of insert only being ligated and those won't transform.
You definitely need 1x ligation buffer in the ligation reaction. Dilute the entire reaction afterwards if necessary to prevent arcing.