Barrick Lab :: ProcedureEvolvabilityMutationAccumulation

---++ 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 12 colonies. These will end up being our 12 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 12 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 12 colonies and spin it in a glycerol well. Store plate in the -80°C freezer.
   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. For each of the 12 plates: pick the last single colony and passage onto a new MG plate with a sterile toothpick. 
   1. Again, streak onto a new MG plate, in a circular fashion, diluting as you go around. 
   1. Repeat this passaging each day. 

---++ Freeze Clones

   1. Every 4 days, before passaging pull out the freezer plate from the -80°C freezer and, in the next row down, add 50&#956;l 80% glycerol to each well. 
   1. Using a sterilized, rounded pasteur pipet, pick each of the 12 colonies and spin it in a glycerol well. Return plate to the -80°C freezer.

---++ VNTR analysis!

*Note to self: Talk to Robin about designing labeled 5' primers, etc!*

| *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
Contributors to this topic
LindseyWolf
Topic revision: r5 - 2011-11-02 - 16:08:23 - Main.LindseyWolf