Troubleshooting Notebook

We are working on creating a troubleshooting manual for others who use MoClo.

Failing Level 1 Reactions

After many of the team’s level 1 reactions failed, we sat down to troubleshoot the situation. We first discussed possible reasons for a reaction failing and decided on the following reasons:

  • Enzyme was not added, was no longer effective, or was the wrong one
  • Part is contaminated
  • Wrong Destination Vector
  • Wrong antibiotic plate used during transformation
  • Buffer no longer effective
  • Forgot to add a part of destination vector
  • Wrong thermocycler settings
  • Competent cells no longer effective
  • Forgot heat shock step during transformation
  • Cell recovery time was not long enough during transformation

We then talked about would could cause blue colonies resulting from a transformation

  • Restriction enzyme did not cut correctly
  • Ligase no longer effective
  • Wrong fusion site design
  • Labeling error so incorrect part used
  • Too much destination vector was added

We also came up with the following reason as to why there could be no growth after a transformation:

  • XGAL which is toxic did not dry long enough on the plate when performing blue-white screening
  • The wrong antibiotic plate was used
  • A toxic gene was used
  • Competent cells no longer effective

After examining the specific level 1 reaction that failed, we were able to find that they each contained B0015_DF. After tracing back when the last time this part was made, it was found that it had been switched with another part during minipreps. We then restreaked out the part and corrected the error.

Terminator B0015_DF

We had many Level 1 MoClo Reactions with B0015_DF fail. We sequenced our existing plasmid stock and discovered that it was mixed up with the RBS B0033_BC. We struck out the glycerol stock from the -80C freezer and sequence analyzed that part. It turned out this part was correct so we replaced the plasmid, -20C, and -80C stocks with the correct B0015_DF.