DNA Assembly (digest & ligate)

LAB PROTOCOL

Plasmid constructs are assembled from individual DNA fragments that contain the desired biological parts of interest. The DNA assembly process produces circular plasmid DNA that is able to independently replicate in E. coli host. The process includes at least two DNA fragments, a vector backbone which incorporates the origin of replication and the antibiotic resistance module, and one or more inserts. Choose a set restriction enzyme (RE) that produce compatible ends (stick or blunt) for vector and insert DNA fragments.

  1. Amplify vector backbone DNA in E. coli host and extract purified DNA by miniprep (or midiprep). Quantify prepped DNA using Nanodrop spectrophotometer.

  2. Digest 2-3 micrograms of vector backbone DNA with chosen REs according to manufacturer specifications.

  3. If insert DNA fragment resides in plasmid construct or obtained by PCR amplification, digest 1-2 micrograms of DNA with chosen REs according to manufacturer specifications. If insert DNA fragment was obtained by PCR and its intended use is for blunt end ligation, remember to add 5’-phosphates by PNK phosphorylation reaction*

  4. Following digestions, carry out a reaction clean-up (kit) to remove excised DNA, proteins and salts. Quantify purified DNA using Nanodrop spectrophotometer. (If excised DNA is longer than 150 base pairs, purify DNA by gel extraction and purification).

  5. Set up a ligation reaction with 50 nanograms vector backbone DNA and insert(s) mass equal to 3x the molar concentration of vector DNA in the reaction (calculator tool: https://nebiocalculator.neb.com/#!/ligation ). Incubate 30 minutes at room temperature for sticky end ligation reactions or 2 hours for blunt end ligation reactions.

  6. Following ligation reaction, de-salt the reaction mix by membrane dialysis (for electroporation transformation) for 20 minutes.

  7. Transform competent E. coli cells using 5-10 microliters of the ligation mix.

*DNA fragments obtained from PCR lack a 5’ phosphate group. PNK phosphorylation reaction was carried out on DNA fragments for the addition of a 5’ phosphate group that would enable the joining of DNA fragments in blunt end ligation reaction. PNK phosphorylation reactions were carried out using 100 ng of purified DNA fragment, 2 μl T4 DNA Ligase Reaction Buffer (10x) and 1 μl T4 Polynucleotide Kinase made to 20 μl total volume using ddH20. The reaction was incubated for 30 minutes at 37°C which was followed by enzyme inactivation for 20 minutes at 65°C. Alternatively, purified DNA fragments from PCR were digested with restriction enzymes to produce compatible ends for stick end ligation reaction.

**DNA inserts from synthesized oligos: Pairs of chemically synthesized oligonucleotides of complementary sequences were annealed to produce DNA fragments with compatible ends for DNA assembly. This method was used when the desired DNA fragments were of small size <100 bp, but long enough to prohibit construction by PCR methods. Reactions were set up using 10 μl of each oligonucleotide of 100 μM stock concentration, 4 μl T4 DNA Ligase Reaction Buffer (10x), 1 μl T4 Polynucleotide Kinase made to 40 μl total reaction volume with ddH2O. The reaction was incubated for 1 hour at 37°C. Next, the reaction volume was made to 400 μl with ddH2O and heated to 100°C for 5 minutes. Finally, the reaction was left to cool to room temperature for 2 hours.

 
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