(r3) ProcedureEvolvabilityMutationAccumulation < Lab

---++ Day 1: Plating the Mixed Population

   1. Find microsatellite-containing strains in the -80°C freezer. They will be labeled with the appropriate microsatellite sequence located in the rhamnose operon (eg. "(CA)<sub>12</sub>"). See below for the list of microsatellites.
   1. Using _careful_ sterile technique, dilute 1:1000 in saline by adding 10&#956;l frozen stock in 10ml saline. 
   1. Dilute this again 1:10 by adding 1ml of the first dilution in 9ml saline. 
   1. Plate 100&#956;l on minimal glucose (MG) plates. Label plates with strain and date. 
   1. Let grow overnight at 37°C. 

---++ Day 2: Picking Colonies

We'll start off picking 24 colonies. These will end up being our 24 replicates that are passaged simultaneously day-to-day.

   1. Get out a sterile 384 well freezer plate. Label with strain and date. In the top row, add 50&#956;l 80% glycerol. _Sterility is very important here._
   1. Choose the 24 colonies closest to the written date on the plate that are far enough away from other colonies to be picked without contamination. 
   1. Using a sterilized, rounded pasteur pipet, pick each of the 24 colonies and spin it in a glycerol well. 
   1. With a sterile loop touch the remainder of the colony on the plate and streak onto a new MG plate, in a circular fashion, diluting as you go around. 
   1. Let grow overnight at 37°C.

---++ Day 3: Passaging Colonies

   1. Pick the last single colony and passage onto a new MG plate. 

---++ Freeze Clones

| *Number* | *Sequence* | *Position* | *Fluorescent Tag?* | 
| 1 | (CA)<sub>6</sub> | _rhaT-rhaD_ | |
| 2 | (CA)<sub>12</sub> | _rhaT-rhaD_ | |
| 3 | (TA)<sub>12</sub> | _rhaT-rhaD_ | |
| 4 | (A)<sub>24</sub> | _rhaT-rhaD_ | |
| 5 | (AGA)<sub>8</sub> | _rhaT-rhaD_ | |
| 6 | (GGA)<sub>8</sub> | _rhaT-rhaD_ | |
| 7 | (G)<sub>9</sub> | _rhaT-rhaD_ | |
| 8 | (G)<sub>12</sub> | _rhaT-rhaD_ | |








-- Main.LindseyWolf - 18 May 2011
Barrick Lab > ProtocolList > ProcedureEvolvabilityMutationAccumulation
Topic revision: r3 - 2011-05-19 - 18:00:21 - Main.LindseyWolf