Preparation      Blunting      Phosphorylation      Purification      Ligation      Troubleshooting

Most cloning experiments involve the insertion of a DNA fragment into a plasmid for downstream protein expression, insertion of a multiple cloning site or the addition of a property to the vector (e.g., a drug resistance marker, a promoter, a signal sequence, etc.). The following tips will help with the design and troubleshooting of cloning experiments.

Preparation of Insert and Vectors

Insert from a plasmid source

• Digest plasmid with appropriate restriction enzymes to produce a DNA fragment that can be cloned directly into a vector. Unidirectional cloning is achieved with restriction enzymes that produce non-compatible ends.
  

• Design primers with appropriate restriction sites to clone unidirectionally into a vector
• Addition of 6 bases upstream of the restriction site is sufficient for digestion with most enzymes
• If fidelity is a concern, choose a proofreading polymerase such as Phusion or Vent DNA Polymerase
  
• Guidelines for PCR optimization can be found here
• Purify PCR product by running the DNA on an agarose gel and excising the band or by
  using a spin column
  
• Digest with appropriate restriction enzyme
  

• Annealed oligos can be used to introduce a fragment (e.g., promoter, polylinker, etc.)
• Anneal two complementary oligos that leave protruding 5´ or 3´ overhangs for ligation into a vector cut with the appropriate enzymes
• Non-phosphorylated oligos can be phosphorylated using T4 Polynucleotide Kinase
  

• Digest vector with appropriate restriction enzymes. Enzymes that leave non-compatible ends are ideal as they prevent vector self-ligation.

• Dephosphorylation is sometimes necessary to prevent self ligation. NEB offers two products for dephosphorylation of DNA:
  
  • Calf Intestinal Phosphatase (CIP) is a robust enzyme that will function under many different conditions and in most NEBuffers. However, CIP cannot be heat inactivated and requires a purification step before ligation. To improve DNA purification, do not use more CIP than recommended.
  • Antarctic Phosphatase (AP) can carry out all the same functions as CIP and can be heat inactivated. It has a strict requirement for zinc but will work in all 4 NEBuffers if zinc is included.

• In some instances the ends of the insert or vector require blunting
• PCR with a proofreading polymerase will leave a predominantly blunt end
• T4 DNA Polymerase or Klenow will fill in a 5´ overhang (e.g., EcoRI) and chew back a 3´ overhang (e.g., PstI)
• Alternatively, the Quick Blunting Kit is optimized to blunt and phosphorylate DNA ends for cloning in less than 30 minutes
• Purify the vector and insert before ligation by either running the DNA on an agarose gel and excising the appropriate bands or using a spin column
• DNA can also be purified using β-Agarase I with low melt agarose or an appropriate spin column or resin
  
• Analyze agarose gels with longwave UV (360 nM) to minimize UV exposure that may cause DNA damage

• For ligation to occur, at least one of the DNA ends (insert or vector) should contain a 5´ phosphate
• Primers are usually supplied non-phosphorylated; therefore, the PCR product will not contain a 5´ phosphate
• Digestion of DNA with a restriction enzyme will always produce a 5´ phosphate
  
• A DNA fragment can be phosphorylated by incubation with T4 Polynucleotide Kinase

• Purify the vector and insert before ligation by either running the DNA on an agarose gel and excising the appropriate bands or using a spin column
  
• DNA can also be purified using β-Agarase I with low melt agarose or an appropriate spin column or resin
  
• Analyze agarose gels with longwave UV (360 nM) to minimize UV exposure that may cause DNA damage

• Use a molar ratio of 1:3 vector to insert
• Thaw and resuspend the Ligase Buffer at room temperature
  
• Blunt ends often require longer ligation or high concentration ligase
  
• The Quick Ligation Kit is optimized for ligation of both sticky and blunt ends
• After ligation, chill on ice and transform
• DO NOT heat inactivate when using the Quick Ligation Buffer as this will inhibit transformation

• To confirm that the ligase, ligase buffer and competent cells are functional, repeat the experiment with an uncut plasmid and a single cut plasmid with and without T4 DNA Ligase
• Repeat with fresh buffer as the ATP or DTT may have degraded
  
• Purify the DNA as the presence of NaCl or EDTA will reduce ligation efficiency
  
• Confirm there is a 5´ phosphate on either the insert or plasmid
  
• Ligation produced only linear DNA because the DNA concentration was too high. Keep the total DNA concentration between 1-10 µg/ml.