cloning problem - (Sep/05/2011 )

Hi everybody. i am struggling to clone my 1.7 kb insert into pet21a. First cloned my pcr poduct insert into topoTA, and then dd it with my re ecor1 and hind III with neb. there won't be any band when PCR8 was digested. i used ecor1hf enzyme and hind III and incubated 3 h at 37. so i am sure it is my insert. and i saw by insert exact place. i gel pured it. i cut my vector with the same enzymes in the same conditions and gel prufied it too. i run the cut vector and the dd vector and see my restriction is ok. i did lots of ligations. with fermentas 1h at 22C, 4 C overnight with different molar ratios. my enzymes are new. i transformed it ino chemicallay comp. cells dh1ob and a supplyers cell top10 which we purchased. i transformed my dd vector and have no colonies. i transformed an empty circular vector and have lots of colonies but when i transformed my ligation, there is no colony. i have another stratagene ligase for 4 years old and neb enzyme for 8 years old. dou you think they will work? hdid anyone try it before? which enzyme and temperature is the ebst for sticky end ligation?could anyone please offer me another way because i am going to out of my mind.

many thanks...

Things you might need to address,
Make sure both your restriction enzyme work.
insert to vector ratio - atelast 4:1
Efficiency of competent cells will always be a major concern when dealing with ligation transformation.
i would suggest you to try overnight ligation at 15 degrees.

thank you very much for your advices. which enzyme do you usually use for ligation?

The one from NEB....

T4 DNA ligase is an unstable enzyme and will denature even at -20C.
I would not use either the 4yr or 8yr vial of ligase.

Also, T4 Ligase buffer does go bad with repeat freeze thaw cycles. The buffer has a strong smell of DTT. If the buffer does not smell it has been oxidized and the ATP is gone, Throw the vial away and get a new vial. For this reason always aliquot the T4 ligase buffer.

Given that your enzymes are new (I am assuming that the T4 ligase used was also new), I think you might want to check the DNA concentration of the gel purified DNA. Just to make sure you do have DNA after gel purification. You use a nanodrop or better yet run some DNA on a gel (use a small well)

Next, check that there is DNA in the tube just prior to transformation. RUn some of the clear up ligated DNA onto a gel in a tinny well (smallest well you can fine). You should see high molecular weight DNA bands, which indicate that the ligation had worked and that you have not lost your DNA.

thank you very much for the offers.

Do you dilute the ligation reaction prior to transformation?

no, i will try this because i am still unsuccessful

Try mixing the insert and vector before gel purification, ligating, and transforming. You will certainly get some colonies without inserts, but will also likely get your insert. Gel purification can often cause more problems than it solves.

I am st'll work'ng on my recomb'nant colony but ' have not get any result I want to ask a quest'on my gene contains signal peptide when i want to clone and express it in pet21a do i have to clone the signal peptide part too or can i clone it without its signal peptide

does it change the enzyme activity