How to Troubleshoot a Three Fragment Ligation

Ligation is a method used in biological research for joining separate strands of DNA. The process has been simplified using commercially available ligation kits. A ligation procedure is generally followed by transformation, a process inserting the joined DNA into a bacterial vector recipient, to clone and amplify the DNA. Due to the complexity of the method, results often do not correspond with the desired product and require troubleshooting. This is more predominant in an attempt to join multiple fragments, such as in triple ligation.

Things You'll Need

• Ligation kit
• Ligation protocol
• Agarose gel
• 50 X TBA buffer (242 g Tris base, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA diluted to 1 liter with water)
• Deionized water
• DNA purification kit

Show More

Instructions

Troubleshoot Transformation

• 1

  Formulate a list of steps involved in ligation and transformation. Troubleshooting any scientific protocol generally begins with the final step, working backward.

• 2

  Compare purified DNA product from the sample to the control DNA, provided with the kit. Once DNA has been transformed and cloned in bacteria, it can be isolated, purified and checked by agarose gel electrophoresis. The results for electrophoresis would indicate whether transformation was successful.

• 3

  Determine bacterial growth during cloning. Most bacterial vectors are constructed so only those with a DNA insert -- will grow on a medium containing ampicillin, or other antibiotic. Poor growth could establish that the DNA sample was not successfully inserted into the vector.

• 4

  Confirm that the ligated DNA sample was purified, prior to transformation. Buffer salts and ligation enzymes could inhibit transformation of the inserted DNA. It is also important to ascertain that the ligation enzyme was inactivated by heat, following completion of the ligation procedure. Active ligase could potentially interfere with transformation.

• 5

  Check the date on bacterial stock used for transformation. Old bacterial cells loose efficiency over time. Eventually the bacterial cells are incapable of transformation and quality cloning. Once the process of transformation has been diagnosed, you may begin analyzing the ligation procedure.

Troubleshooting Ligation

• 6

  Confirm the purity of your DNA sample, prior to ligation. Salt contaminants in the DNA sample could result in poor ligation. The DNA sample should be purified and free of salts and enzymes, before it is used in ligation.

• 7

  Confirm whether the ends of DNA strands to be ligated are blunt or sticky. Ligation is used to link separate strands with blunt ends or sticky ends, following digestion with designated restriction enzymes. T4 DNA ligase is the enzyme of choice for blunt end DNA -- but it will also work for DNA with sticky ends. Other DNA ligases may only work for DNA, following a restriction digest to create sticky ends. It is important to use the proper ligase for the DNA tested.

• 8

  Check concentration of DNA samples before ligation. Using DNA with high concentration often causes DNA to become linear. The kit instructions will provide information on the correct amount of DNA to use in a ligation reaction.

• 9

  Replace ligase enzyme with a new lot. Enzymes are particularly sensitive at room temperature and continued freeze-thaw cycles. The ligase could be damaged following a number of uses, simply by freezing and thawing too many times. Some kits recommend that ligase be aliquoted into smaller portions during initial use, to reduce the number of times it is re-frozen.

• 10

  Check the ligation kit. Often the problem with ligation is using a kit that has expired or been contaminated with other DNA and salts.