problem in ligation and transformation - (Apr/25/2006 )

Hi, I want to ligate 1500 bp pcr product in to T vector but I didn't succeed . I have checked compotent cell and it grows in LB agar without Amp.
I even transform the pcr fragment of the kit and it was successful. my compotent is DH5 . I use Fermentas kit.
I appreciate your kindness.

could you please provide us more details of your protocol?

The fact that the cells are viable does not make them competent...

As aimikins says, can you give us more detail of your experiment? What procedure did you use to produce competent cells? How did you do your ligation and transformation? Did you digest your vector and insert with a restriction enzyme(s), or is this a TA cloning? If this is restriction digest ligation, what enzymes did you use? Did you engineer restriction sites into your primers, or are you using sites within the product? How did you digest and prepare your PCR product for ligation (i.e. did you gel-purify your resticted fragment)? Did you do a circular vector transformation to check the competency of your cells?

Hi Guys

I'm really having problems with my ligation reactions! I've got a 1kb insert which is a PCR product with different REs on the ends. It is not phosphorylated.

I've cut my 5.4kb plasmid with the same 2 REs as my PCR product but since my insert is not phosphorylated, the plasmid has not undergone SAP or CIP treatment. So far, no success with my ligations.

Have run positive and negative controls which are both OK, so looks like the transformation step and the competent cells are working.

Have people had better success by using a phosphorylated insert with a dephosphorylated vector in their ligation reactions?

I ran a ligation efficency gel using 2ul of my ligation reaction, to see if there was a heavier band but couldn't see any bands - do I need to load more of my reaction onto the gel or am I not using enough DNA in my ligation reactions?

Am getting frustrated as this is a month I have been working on this! Thanks!

It could be your vector/insert didn`t get cut properly. I have problems to cut pCDNA3.1 with EcoRI and BamHI f.ex.
You can phosphorylate your insert and dephosphorylate the cut vector to minimize the background.
By the way, I was told once that if you cut your insert with the REs, it is phosphorylated afterwards.

Oh! A-ha! (light bulb comes on!) That may be the problem - didn't think about the fact that once I digest my PCR product with REs, the ends are now phosphorylated!

I was just thinking about the original ends which were not phosphorylated to being with!!! So obviously, in this case I would have to dephosphorylate my vector! D'oh!

Thanks chalet2!!!! I owe you a beer!

I've already ordered some CAP and SAP so will wait for those to arrive and dephosphorylate my vector before trying again. Guess the transformations I did today won't work! Oh well!

it may have worked; you might wish to miniprep a few and see

you don't have to dephosphorylate with different ends; it just reduces possible background from single-cut vector in the mix

My two ends are different REs.. so plasmid has been cut with 2 different cut sites..

Aimikins - do you think it could have worked? I don't see how, since my vector is phosphorylated and my insert is phosphorylated too....?

I wouldn't mind going ahead and finishing up the transformation protocol if there is the chance that my ligation may have worked. I did run a ligation efficiency gel and thought I saw some bands higher up than my wildtype plasmid but that could just be the gel running funny?

sure it could have worked

the only time it is a no-go is when both vector and insert have been dephosphorylated

I cant' answer your second question; I rarely run gels of my ligation product because I think you can see most of what you need to about how the reaction went when you see your plates the next morning, and you frequently seem to end up with DNA from outer space...also, you don't need very much DNA to transform. If you were getting big strong bands from that gel, how much DNA are you transforming/in what volume? perhaps you are adding too much and that is the problem?

The bands are very faint but I have a gel doc system so I crank up the exposure time to about 5 seconds and then I can see the bands. The ligation volume was 20 ul overall because I needed about 10ul if insert for most of my ligation rxns... I lose a lot of DNA with the purificatio steps, even when I use Qiagen's QiaQuick kit which they claim recovers 80-90%

Ok, I've gone ahead and heat shocked my cells so will plate and see if I get any colonies tomorrow.

Wish me luck!