Troubleshooting
Having problems with common lab protocols–measuring OD600 values, making media and plates, microscopy? We’ve compiled a list of common problems and fixes for your troubleshooting.
Troubleshooting
For the first 3 Secretion Assays, the OD600 Readings recorded were not multiplied by 5 to account for the 1:4 dilution of the overnight culture samples in the cuvettes. This meant the cultures were actually being induced when the cultures were well into the death phase of bacterial cell growth. This meant that bacterial cells were in the process of lysing, which is why we saw decreased expression of the mEGFP from these samples in the Western Blot.
For the rest of the secretion assays, we accounted for the 1:4 dilution of the cultures in LB Media and multiplied those values by 5.
When bacteria are taken from glycerol stock and are immediately placed into the induced LB medium, they tend to not grow as extensively aa they do in LB medium without inducer. For aggregation assay, we needed a consistent OD600 to begin with induced bacteria often grow very little over night resulting in sizeable difference between the noninduced and the induced cultures. Instead, grow overnight cultures in plain LB media one night before and then sampling 1:100 dilution of them into LB media with inducer results in a better growth of the bacteria.
When the bacteria are not on shaker, they slowly settle down in the tube and form a gradient. It is difficult to retain a uniform bacterial density or OD600 value for the culture. Our fix to this problem was to dilute the original sample into a new culture tube with a target OD600 value. This enables experimentation with the bacteria without having to remeasure the the OD600 value for short periods of time.
For microscopy sessions, mammalian and bacterial cell integration would often require a MOI of 100 or 10. However, the each well does not have large number of mammalian cells and very little volume of bacterial cells are needed to obtain the intended MOI especially if the bacterial cultures have OD600 that exceeds 1.00. Since qualities less than 2 µg were not precise, double dilutions of the bacteria was implemented such that the second dilution provides precise measurement of the number of bacterial cells for being added to the mammalian cells.
We would find ourselves in the situation having to run out of plates while completing transformations. Making an assumption for the plate LB agar medium being 30 mL, a 1:1000 dilution of the antibiotic can be used to prepare a plain agar plate. For example, if there is 30mL of media in the plate then you plate 30uL of antibiotic, spread it with the plating beads, then let it dry before you use the plate.
We would find ourselves in the situation having to run out of plates while completing transformations. Making an assumption for the plate LB agar medium being 30 mL, a 1:1000 dilution of the antibiotic can be used to prepare a plain agar plate. For example, if there is 30mL of media in the plate then you plate 30uL of antibiotic, spread it with the plating beads, then let it dry before you use the plate.
Maintain a detailed text file along with microscopy data containing times when things were added/removed, what order the wells were imaged in, what kind of imaging was done, what was in the wells.
It is easy to lose track of which samples are which.
Once the medium for making agar plates has been removed from the autoclave. Do not wait to pour the medium into the plates. Waiting too long will cause the agar to start settling to the bottom in the media. This causes the medium poured from the top of the jar to not solidify well (little to no agar) while the medium at the bottom of the jar solidifies rather quickly (contains a lot of agar). If ever encountered with the situation where the autoclaved medium becomes to cool, collect the liquid portion in different material and use the agar-rich portion of the medium to make plates. This will avoid of wastage of plates that otherwise would not solidify.
Lesson Plan
Use this worksheet as a guide to SnapGene and Gibson Assembly.
