NEB PCR Cloning Kit (NEB #E1202/E1203) Tips

  1. Compatible with other strains: NEB10-beta cells (NEB #C3019) (which are included in the E1202 kit) are recommended for best results. However, you can use other strains of high-efficiency competent E. coli, as long as they grow quickly. NEB Stable Competent E. coli (NEB #C3040) is an excellent choice for cloning direct and indirect repeats, and works well with the PCR Cloning Kit. NEB Turbo Competent E. coli (NEB #C2984) and NEB Express Competent E. coli (NEB #C2523) are also good choices. NEB 5-alpha Competent E. coli (NEB #C2987) grows slowly, so the number of background colonies is higher than with NEB 10-beta Competent E. coli (NEB #C3019).
  2. Compatible with blunt or single-base overhangs: You can use the NEB PCR Cloning Kit to clone any fragment that has a blunt end or a single-base overhang.
  3. Use an insert:vector ratio of 3:1: A higher insert:vector ratio can actually result in fewer colonies. This is due to the fact that inserts may ligate to both ends of the vector, which will prevent the cloning of your amplicon insert.
  4. Plate 50 µl or less of the 1 ml outgrowth. Plating too much of the outgrowth can increase background, and cause problems with colony PCR. If you need more colonies, spread 50 µl of outgrowth onto each of multiple plates. If you have used a 15 minute ligation time, also plate 50ul of a 1:10 dilution of the outgrowth.
  5. Follow the protocol: The protocol has been highly optimized to have a low background; if you have inadvertently deviated from the optimized protocol (e.g., extended ligation incubation, overly-concentrated outgrowth), compensate by plating less outgrowth (< 50 µl).
  6. Important to stop ligations: If you wish to store your ligations to allow transformations at a later time, make sure your freezer is cold enough (- 20°C) to freeze the ligations. Or, you may quick freeze with a dry ice/alcohol bath before transferring the samples to -20°C. If you find your freezer-stored ligations have remained in liquid form, this may have allowed further low-level ligation of the vector backbone to occur. In this circumstance, plate less outgrowth than the standard 50ul, ie 50ul of a 1:10 dilution.
  7. Follow the transformation protocol carefully. The number of colonies will decrease significantly if you incubate the ligated DNA with the competent cells for less than 20 minutes, or if the outgrowth with SOC is less than 60 minutes.
  8. Do not incubate the transformation plates at room temperature. The slow growth rate of the cells at room temperature will increase the number of background colonies.
  9. Add the cloning mixes 1 and 2 (which can be mixed together for the day's experiment) to the reaction last. Some people try to save time by preparing a mix of water, ligation master mix and pMiniT, aliquoting this to tubes and adding the insert DNA. This allows pMiniT to recircularize, since ligation can begin before the amplicon is added, and this may result in lower cloning efficiency.