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Common Pitfalls

This section of the tutorial covers common errors made in the lab that are easily avoided.

Using contaminated media

Arking JCATutorialContamPlate.jpg

A common error is rescuing transformations in contaminated media. The plate at right shows two colony morphologies. The minor population are white E. coli colonies, the majority are yellowish. They came from a bottle of 2YT media that had visible cloudy streaks. Before you use growth media for picking colonies or rescuing a transformation, check to make sure the media has no cloudiness or floating debris. You can't rescue contaminated media, so throw it out. Along these lines, it is important when making media that you autoclave it without significant delay. The powder in the media bottles is not sterile, and will readily grow as soon as you add water. This growth will alter the composition of the media. So, it is important that you kill that stuff by autoclaving before it gets a chance to amplify. Waiting 1 or 2 hours to autoclave is ok. Waiting overnight is fatal.

Storage methods for E. coli

There are many ways to store E. coli including liquid cultures, agar plates, agar stabs, agar slants, and -80 cultures. It is important to know how long these things stay viable. Liquid cultures growing at 37 degrees are viable for approximately 48 hours. After this time (at 37), the bacteria will be mostly dead, and even if they aren't dead will likely have lost plasmids. If you take liquid cultures out of the incubator and place them on the bench at room temp, they will be reasonably stable for a few more days. If you put a saturated liquid culture in the refrigerator, it will last at least a week. Never directly freeze media at -20 or -80 without adding a cryoprotectant such as glycerol or DMSO. They are not stable without it. However, if you do add DMSO (~10%) or glycerol (8 to 25%), cultures of E. coli in pretty much any media (M9, LB, SOB, PBS buffer, etc.) coming from any type of growth (any growth phase of liquid growth, scraped off a plate, pelleted from liquid cultures and resuspended in media) are stable for years stored at -80. Many people will flash freeze these stocks before placing at the -80, but it is perfectly ok to simply drop the samples into the -80 and let them slow-freeze. Agar plates are generally stable stored at 4 degrees as long as they are preventing from drying and contamination by wrapping in parafilm. They can last as long as 2 months if you're lucky. Agar stabs and agar slants involve storing bacteria on the surface or embedded within agar in sealed tubes. They are stored at room temperature and are stable as long the agar does not dry out. They can last years but are generally not as stable as -80 stocks.

Finding oligos from your oligo inventory

Let's say you need a sequencing oligo that anneals within your gene of interest, and you know you've done this before, but you don't remember the oligo name. Assuming you've maintained an "all oligo" style spreadsheet, you're in luck. *Go to the pairwise alignment tool at [1].

  • paste the gene of interest sequence into the first box
  • Highlight the column of sequences in the all oligo spreadsheet, copy it, and paste in the second box
  • hit submit

This will only give you one good forward and one good reverse oligo at a time, but you can iteratively go through the list by deleting each one you find.