Profiling Ribose Operon Deletions

Day 1: Search strain databank for desired population.

  1. Login to lab website, open lab databases.
  2. Search under "strains" for A-5 or A-1 (Ara-5 or Ara-1).
  3. Early generations (mixed populations will show up first, using the number to find it in the -80C freezer.

Plate on minimal glucose (MG) plates.

  1. Using careful sterile technique, dilute frozen stock 1:1000 so 10μl in 10ml saline.
  2. Plate 3 different concentrations as the stock concentrations vary widely (eg. 500 gen Ara-5 should be plated from this first dilution, but 500 gen Ara-1 must be diluted an additional 1:100). So to make 103, 104, and 105 dilutions, from the first 103 dilution add 1ml to 9ml saline and 100ul to 10ml saline. Plate 100ul of each dilution onto a separate plate. Label each with date and dilution.
  3. Grow overnight at 37C.

Day 2: Picking colonies.

  1. Using autoclaved polypropylene 96 well deep well plates, add 1ml LB to each well. (This should be a large enough volume to get more concentrated gDNA later).
  2. Using a sterile loop (keep sterilizing in flame after each colony), pick up to 95 colonies (leave at least one well for a blank). We'll be picking colonies "randomly" so on the plate with a dilutions with the most distinct and easily picked colonies, take colonies closest to where the date is written on the plate and work outwards.
  3. Grow overnight at 37C.

Day 3: Freezing samples.

  1. In a polypropylene 96 well storage plate add 40μl sterile 80% glycerol to each well. Add 200μl over overnight LB culture to each and pipet up and down to mix. If any wells from the overnight didn't grow, mark those on the storage plate. Label the storage plate and transfer to -80C.

Genomic DNA isolation (taken directly from Invitrogen protocol).

  1. Heat up water bath to 55C.
  2. Centrifuge remaining 800μl in deep well block >2,100 x g for 10 minutes.
  3. Follow the rest of the protocol on p14.
  4. Once lysis is complete continue to p21.
  5. Follow protocol on page 21 (Be sure to use wash buffer 1 first and wash buffer 2 for the second wash. Also, use gloves when working with guanidine HCl.
  6. Elute using 200μl elution buffer.
  7. Store at -20C until ready for PCR.

Day 3, 4, or later: PCR ribose operon.

  1. Nanodrop several of the wells to make sure they are similar concentrations.
  2. Set up PCRs for the enture ribose operon using primers 1 and 19 (see specifics for PCR below).
  3. Use "PCR long" program in thermocycler. This will take ~6hr so doing this overnight is often most convenient.

Specifics for Ribose Operon PCR Protocol

  • To get 300nM in a 50μl reaction add 0.6μl and 0.8μl for primers 1 and 19, respectively... multiply that by number of reactions.
  • Use 1.7μl dNTPs (longer products require more nucleotides).
  • Use 1μl template gDNA (this is subject to change).

Run gel.

  1. Use a 0.5% agarose gel with SYBR Safe (5μl/100ml SB buffer).
  2. Image ("Protocol 1" on imager).
  3. If you can't see a band it's possible that it's too large to PCR properly (eg. the ancestor is 9kb). If you don't see a band, repeat PCR with primers 1 and 20. If there is not deletion in the ribose operon, you should see a 4.5kb band with these primers, corresponding to the portion of the operon which would be deleted by 2000 generations. Run a gel with this PCR product and image to verify a lack of deletion.

Strain Generation Primers μl/50μl rxn Product Size (kb)
Ancestor 0 1 & 19 0.6/0.8 rbs operon 9.01
Ara-1 2K 1 & 19 0.6/0.8 rbs operon 2.07
Ara-5 2K 1 & 19 0.6/0.8 rbs operon 3.38
Ancestor 0 1 & 20 0.6/0.6 rbsD-rbsB 4.5
Ara-1 2K 1 & 20 0.6/0.6 deleted
Ara-5 2K 1 & 20 0.6/0.6 deleted

Note to self: Include primer positions

-- Main.LindseyWolf - 18 May 2011

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