Hi,

Here goes the protocol,

Amount of PCR product to use:

A 1:1 molar ratio of vector:insert produces the best ligation efficiency. In general, 0.5 to 1.0 μl of a typical PCR sample with an average insert length (400-700 bp) will give the proper 1:1 molar ratio of vector:insert. You may also perform a second ligation reaction using a 1:3 molar ratio of vector:insert if you are concerned about the accuracy of your DNA concentrations.

Do not use more than 2-3 μl of the PCR sample in the ligation reaction as salts in the PCR sample may inhibit the T4 DNA Ligase

Ligation Reaction:

Set up the 10 μl ligation reaction as follows:

Fresh PCR product (Determine how much you need); 10X Ligation Buffer............ 1 μl; pET SUMO vector (25 ng/μl)............2μl; T4 DNA Ligase (4.0 Weiss units) Final volume....... 1μl; Add sterile water to make total volume of 10 μl;

Final Volume (Total) should be of 10 μl

3. Incubate the ligation reaction at 15 degree C for a minimum of 4 hours (preferably overnight). You may also incubate your ligation reaction at room temperature for 30 minutes, if desired. Proceed to transformation into Competent cells.

Note: You may store your ligation reaction at-20°C until you are ready for transformation.

Best wishes!

Thank you sharmin for your answer. Can you tell that is there any difference in protocol for large inserts as >1.5kb ?

Hi Akshay, basically there is NO difference except it might get harder with inserts>10kb or so. Other than that, you should be just fine :)

thanks i will try that.