The Rapid DNA Ligation Kit contains all reagents necessary for ligation. The kit enables DNA ligation with either blunt or sticky ends at 15-25°C. Depending on the DNA concentration in the reaction, the ligation products will be either circular (if the DNA concentration is low) or concatemeric (if the DNA concentration is high). Ligated DNA is suitable for direct use in transformation experiments.
To perform a successful cloning experiment, use these handling steps as guidelines for using the Rapid DNA Ligation Kit (11635379001).
Note: Cloning experiments involve a wide range of products. The ligase enzyme in the Rapid DNA Ligation Kit is only one of them. For success, the entire cloning workflow must be optimized, using appropriate controls for each step.
Note: For plasmid vectors do not exceed 200 ng of DNA in 20 mL reaction volume. Otherwise highly concatemeric ligation products will be obtained and circularization will be suppressed.
The table below gives a typical result.
These values were achieved using competent E. coli JM83 cells prepared according to the Hanahan method. Only 1/10 of the ligation mixture was used for the transformation.
Hanahan cells are sensitive to excess amounts of DNA, i.e., if the whole ligation mixture was transformed, the number of transformants was reduced:
Calcium chloride-treated cells have a higher tolerance for excess amount of DNA.
Note: For cloning in λ-vectors a high amount of DNA should be used to favor formation of concatemers.
There are several different methods to prepare competent E. coli cells. Protocols differ in the grade of difficulty and the reagents used and transformation efficiency achieved. Two of the most common methods are discussed briefly below.
E. coli cells are grown to the early to intermediate log phase and then incubated on ice in the calcium chloride buffer. The maximum efficiency is 1 x 106 to 1 x 107 transformants/per microgram of DNA. To achieve maximum efficiency in any transformation experiment use the following guidelines:
– Always keep the cells on ice.
– Prechill buffers on ice.
– Use chilled (-20 °C) pipettes (glass or plastic), tips and centrifuge tubes.
– Always store bacteria on ice once they had contact with the transformation buffer, even during transport to the centrifuge.
– Chill centrifuge to 4 °C.
– Either use competent cells directly or store at -70 °C in the presence of 30-50 % glycerol. Cells tolerate about half a day storage on ice without dropping of efficiency.
– Note that the activity of Rec A minus strains decreases during storage.
– Use extremely clean glassware and plastic ware.
The Hanahan method uses a more complicated buffer and it is important to have reagents of very high purity. In particular, the DMSO is critical. Basic handling is the same as the calcium chloride method. Transformation efficiencies achieved are in range of 1 x 107 to 1 x 109 per microgram of DNA. As already stated, the purity of the reagents is critical. Only 1/10 volume of the ligation assay should be used for transformation. These cells are very sensitive to excess amounts of DNA.
It is extremely important to perform appropriate controls as follows:
Strains that are used in M 13 cloning have a special genetic construction of an F' plasmid to make sure that the F-pili which are the receptors for M 13 are expressed. Respective strains should be grown on minimal medium or dependent on the strain construction on antibiotic containing medium before inoculation for transformation to make sure that the strains still contain the F' plasmid. For the transformation it is important that the strains are in the early to intermediate log phase. If the respective strain is in the late log phase or stationary phase the F-pili could have been lost to a certain extend so that no receptor would be present on the surface of the cells and the obtained plaques could be very small.
For the Rapid DNA Ligation Kit, we guarantee > 1 x 106 transformants per μg religated pUC18 vector (sticky end as well as blunt end), i.e., this means that only 2.96 pg of vector (1 molecule of pUC 18 = of 2,96 ag [attogram!]), have be to be religated to result in this yield in transformants. This rate of religation is easily achieved in 5 minutes with the Rapid DNA Ligation Kit.
Note: This also means that when ligation products are checked using an agarose gel, a substantial proportion of the vector DNA used will still show up â€žunligatedâ€œ. Considering the above mentioned molecular weight of pUC 18 this is is easily visualized.
The rate and kinetics of concatemer ligation in cloning with λ-vectors are totally different from plasmid ligation. For ligation of concatemers ligase has to connect linear fragments in the presence of high amounts of DNA. For plasmid cloning an insert must first be ligated to one end of the linearized plasmid-vector, and then the other end of the same plasmid-DNA must be connected to the insert.