Quick 3hr Antibiotic Rescue Verification

Overview

This short protocol describes a simple same-day verification of antibiotic removal/"rescue" from counter-selection plates (e.g., AZT). Typically, rescue transformations are verified by PCR at the antibiotic cassette rescue loci, but sometimes this needs to be performed in >10 colonies due to low efficiency transformations or leaky counter selection media where scape mutants are frequent. Therefore, the following quick test can be performed prior to PCR confirmation, avoiding time-consuming PCR checks and limiting failed PCR verifications.

Materials

• Colonies from counter-selection plate
• Small 5mL tubes (Falcon Polystyrene Round-Bottom Tube)
• Sterile (new box) P10/P20 tips

Procedure

1. Set 250uL media with the antibiotic to be tested in a new tube
2. Lightly touch a colony with a sterile tip and make sure some cells are visible on the tip. If colonies are large, avoid transferring a large clump of cells, all you need is a slightly (barely) visible amount.
3. Drop tip with cells into tube.
4. Set to incubate at 37C and 100-200 rpm.
5. 3-4 hrs later check if growth or no growth.

Tips

• It's highly recommended you label each tested colony on the counter-selection plate (e.g., a, b, c, d...etc.). This will allow you to keep the plate, locate and re-use the assayed colonies.
• If the colonies are big enough, simultaneous incubation can be made as in steps 1-5 but without adding the antibiotic, so you have a liquid culture ready for downstream use or PCR verification.
• It doesn't matter whether the 5mL Falcon tube caps are tighly sealed or not. Barely any evaporation happens within 5 hr period and aerobic growth will still be possible even if sealed during this short time. After removing the tubes from the incubator, you can tighly seal their caps to prevent them from evaporation and keep the results visible for days to come.
• If you transfer a large clump of cells to your test tube, it could cause enough turbitity in the low 250uL media amount to confuse your result. You may conclude wrongly that there was growth. Just make sure to use a "barely" visible amount of cells, if not, you'll probably need to re-test if uncertain by re-inoculating from the test tube into another tube with antibiotic in it.

-- Main.GabrielSuarez - 22 Mar 2018