Rapid DNA Ligation Kit Protocol

Introduction to the Rapid DNA Ligation Kit

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.

DNA Ligation Kit Protocol

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.

DNA Ligation Preparation

• Vector/Insert DNA Purity: Use highly purified DNA: We recommend using the High Pure PCR Product Purification Kit or cesium chloride gradient purification. Miniprep DNA may contain impurities that lower ligation efficiency.

• Vector/Insert Preparation: Restriction enzymes should not contain nuclease contamination. Star activity should also be avoided. When double digests are performed with enzymes prone to star activity, consult the Roche Restriction Enzyme Poster (or the Roche Applied Science Catalog) to select the optimal buffer for a given enzyme combination. Whenever possible use Buffer H for double digests.

• Dephosphorylation: Dephosphorylate the vector DNA (except for recircularization), to prevent self-circularization. Follow exactly the instructions for using alkaline phosphatase, in particular for the inactivation step. Alkaline phosphatase must be inactivated in the presence of EGTA and heating for 10 min to +65 °C, followed by phenol treatment and EtOH precipitation. Other procedures including spin columns are not effective. Alkaline phosphatase is an extremely stable enzyme and the treatment described in the package insert is for complete inactivation.

• DNA Storage Buffers: A DNA dilution buffer is provided with the Rapid DNA Ligation Kit. Use this buffer to adjust the DNA concentration for the ligation. Always resuspend DNA in buffers not containing more than 0.1 mM EDTA. Higher EDTA concentrations will inhibit ligase activity, because EDTA complexes the Mg²⁺ ions that ligase requires as a cofactor. The best option is using a Tris-buffer with 10 to 50 mM Tris; pH 7.5 to 8.

Ligation

• DNA Buffer: To dissolve DNA to be ligated, use the DNA dilution buffer provided with this kit. Alternatively, dissolve DNA in a buffer either with very low EDTA concentration up to 0.1 mM or even better without EDTA.

• DNA Amount: The maximum amount of DNA to be ligated in 5 min should not exceed 200 ng.

• Reaction Volume: For Rapid DNA ligation, the reaction volume is limited to a total of 20 μL. For larger volumes, the reaction time has to be prolonged up to 30 minutes! The volume of all the other reagents in the reaction must be increased accordingly.

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.

• Ligation Buffers: When working with the Rapid DNA Ligation Kit, it is essential to mix the contents of vials 1 and 2 thoroughly directly prior to use.

• Inactivation of T4 DNA Ligase: In principle, T4 DNA Ligase can be inactivated by a 10 minute incubation at +65 °C. Perform this heat inactivation step only, if the ligation mixture is used in experiments other than transformation. Heat inactivation of ligation mixtures prior to transformation/ packaging leads to a drastic decrease (> factor 20) in the yield of transformants/plaques.

• Storage of Ligation Mixtures: Store ligation mixtures at -15 to -25 °C when transformation is performed later.

• Molar ratios of Vector: Insert (plasmid vectors): For the Rapid DNA Ligation Kit it is recommended using molar ratios of vector to insert of 1+1, 1+2, 1+3 or even 1+5 when sticky end ligation is to be achieved. With blunt end ligation the efficiency dropped if higher ratios than 1+3 are used.

The table below gives a typical result.

Ratios sticky	End ligation blunt	End ligation
1+1	3.6 x 106	2.4 x 106
1+2	3.3 x 106	3.0 x 106
1+3	6.8 x 106	1.5 x 106
1+5	1.8 x 107	7.6 x 105

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.

• Amount of DNA in 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:

Ratios	Sticky end ligation	Blunt end ligation
1+1	2.6 x 106	3 x 106

Calcium chloride-treated cells have a higher tolerance for excess amount of DNA.

• Cloning into λ-vectors: For cloning into λ-vectors, a molar ratio of 8 + 1 has been found to be optimal.

Note: For cloning in λ-vectors a high amount of DNA should be used to favor formation of concatemers.

Transformation

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.

• Calcium Chloride Method: This was the first method published for making E. coli cells competent for foreign DNA uptake. It is also the simplest method because it only uses calcium chloride buffer.

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.

• Hanahan Method

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.

Controls

It is extremely important to perform appropriate controls as follows:

• Transformation of uncut vector DNA (e.g., 50 picogram). Result shows transformation efficiency of cells per microgram of DNA.
• Transformation of linearized unligated vector DNA Completeness of restriction enzyme cleavage.
• Transformation of religated dephosphorylated vector. Result shows efficiency of dephosphorylation.
• Transformation of linearized and recircularized vector. Result shows efficiency of ligation.
• Transformation without DNA e.g., only with the DNA buffer. Result shows control for competent cells, i.e., no growth indicates that the cells are not contaminated or already contain a plasmid. Growth on the respective selective medium indicates that the cells are contaminated and contain a plasmid already.

Guidelines for M13 Cloning and Transformation

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.

Important Information for Rapid DNA Ligation into Plasmid Vectors

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 „unligated“. 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.