TITLE:  Copying Archived LTEE Samples 
AUTHORS: Zachary D Blount, Jeffrey E Barrick

[DESCRIPTION]
This protocol describes how to replenish the frozen "fossil" record of the E. coli Long-Term Evolution Experiment (LTEE) by making copies of freezer stocks. Different methods are used to copy population samples versus clonal samples. For the former, the goal is to make a representative copy that maintains the genetic diversity that is present in the population. For the latter, the goal is to make a copy of a homogeneous cell population without allowing it to evolve new mutations.

The culture media and supplies needed for this protocol are described here:
 


[STEPS]
SECTION: Copying Archived Population Samples
1.2. Prepare an autoclaved 50-mL Erlenmeyer flask capped with a 20-mL beaker for each sample that will be revived, plus a blank. Fill each flask with 9.9 mL of DM1000. 




SECTION: Copying Archived Population Samples
1.4. Vortex each freezer vial and pipette 120 µL of the thawed stock into one of the flasks. Wipe off the micropipettor with a Kimwipe moistened with 70 % (v/v) before pipetting from each thawed stock to prevent cross-contaminating the archived samples.
 



SECTION: Copying Archived Population Samples
1.3. Retrieve vials of frozen stock from –80°C storage, place in ice in an insulated ice bucket, and allow the stock to thaw completely.
SECTION: Copying Archived Population Samples
1.5. Incubate flasks       



SECTION: Copying Archived Population Samples
1.9. Dilute the grown cultures 1:40 by pipetting 250 µL into a test tube filled with saline. 
 


SECTION: Copying Archived Population Samples
1.11. Incubate the flasks    for  1440 min .


SECTION: Copying Archived Population Samples
1.7. Prepare another set of the same number of flasks. Fill each flask with 9.9 mL of DM25.

SECTION: Copying Archived Population Samples
1.12. Add 2 mL of 80 % (v/v) glycerol to each flask. Swirl or gently vortex to mix. Pipette the culture-glycerol mix into glass vials that have been labeled with the appropriate sample information. A small glass vial (0.5 dram) can be filled with 1.25 mL each, while large glass vials (2 dram) can be filled with 6 mL each. Be careful not to overfill the vials, or they can break as the contents freeze and expand.

SECTION: Copying Archived Clonal Samples
2. Use this procedure to make copies of samples of clonal isolates from the LTEE.

 

0.23. Fill each flask with 9.88 mL ml o

SECTION: Copying Archived Population Samples
1.8. Prepare one test tube filled with 9.75 mL of sterile saline for each sample.
SECTION: Copying Archived Population Samples
1.10. Thoroughly vortex each test tube three times before using a micropipettor to transfer 100 µL from it into one of the new flasks filled with DM25.

SECTION: Copying Archived Population Samples
1. Use this procedure to make copies of archived population samples from the LTEE that are as close as possible to exact copies of the original samples. 

 

SECTION: Copying Archived Clonal Samples
2.2. Prepare an autoclaved 50-mL Erlenmeyer flask capped with a 20-mL beaker for each sample that will be revived, plus a blank. Fill each flask with 10 mL of DM1000. 
SECTION: Copying Archived Clonal Samples
2.3. Retrieve vials of frozen stock from –80°C storage, place in ice in an insulated ice bucket, and allow the stock to thaw completely.
SECTION: Copying Archived Clonal Samples
2.4. Vortex each freezer vial and pipette 12 µL of the thawed stock into one of the flasks. Wipe off the micropipettor with a Kimwipe moistened with 70 % (v/v) before pipetting from each thawed stock to prevent cross-contaminating the archived samples.

 

SECTION: Copying Archived Clonal Samples
2.5. Incubate the flasks       
SECTION: Copying Archived Clonal Samples
2.6. Add 2 mL of 80 % (v/v) glycerol to each flask. Swirl or gently vortex to mix. Pipette the culture-glycerol mix into glass vials that have been labeled with the appropriate sample information. A small glass vial (0.5 dram) can be filled with 1.25 mL each, while large glass vials (2 dram) can be filled with 6 mL each. Be careful not to overfill the vials, or they can break as the contents freeze and expand.
SECTION: Copying Archived Population Samples
1.1. Disinfect the bench with 70 % (v/v) or 10 % (v/v) and light a Bunsen burner to create an updraft, which will reduce the possibility of contaminants falling into materials during work.
SECTION: Copying Archived Clonal Samples
2.1. Disinfect the bench with 70 % (v/v) or 10 % (v/v) and light a Bunsen burner to create an updraft, which will reduce the possibility of contaminants falling into materials during work.
SECTION: Copying Archived Population Samples
1.6. Disinfect bench with 70 % (v/v) or 10 % (v/v) and light a Bunsen burner to create an updraft, which will reduce the possibility of contaminants falling into materials during work.