Day 1

3. Day 1 1. 1 minute max speed 3 ml 2. 3. Incubate at 37oC overnight Re-suspend 100 µl Incubate at 37oC overnight 4. 100 µl 10-6 Incubate at 37oC overnight 100 µl

4. Day 1 1. Transfer 3 ml of original E. coli culture to a microfuge tube 2. Spin microfuge for 1 minute at max speed 3. Decant supernatant 4. Re-suspend pellet in 100 µl sterile broth 5. Transfer and spread content onto a TBAB + Strep plate • Spread 100 µl of 10-6 diluted original E. coli onto two TBAB plates • Incubate all three plates at 37oC overnight

5. Day 2 1. Incubate overnight

6. Day 2: Determining if mutant is strep resistent, intermediate, or dependence • Count colonies on all three plates • With sterile toothpick transfer colonies from TBAB + Strep plates to a new TBAB + Strep Plate and then a TBAB plate • Incubate plates overnight

7. Day 3: PCR 1. 5. 22 µl 2. 1 µl 1 µl 3. 25 µl of 2X PCR mix • 95° for 5 minutes • 30 cycles of • 95° for 1 minute • 55° for 1 minute • 72° for 1 minute • 72° for 10 minutes 1 µl 100 µl 4.

8. Day 3: PCR • Add 22 µl of sterile water to 25 µl of 2X PCR mix (contains nucleotides, buffer, and Taq polymerase) • Add 1 µl of 10 µM Primer 1 (up) • Add 1 µl of 10 µM Primer 2 (down) • Add 1 µl of suspended E. coli • Place tube in thermocycler • 95° for 5 minutes • 30 cycles of • 95° for 1 minute • 55° for 1 minute • 72° for 1 minute • 72° for 10 minutes

9. Day 4: Machery-Nagel PCR Cleanup Protocol 1. all Centrifuge column for 1 minute at 11,000 Xg 100 µl 2. 3. Centrifuge column for 1 minute at 11,000 Xg 700 µl 4. Centrifuge column for 2 minutes at 11,000 Xg

10. Day 4: Continued 5. = 6. 25 µl Centrifuge column for 1 minute at 11,000 Xg sit at room temperature for 1 minute 7. DNA

11. Day 4: Continued 8. 10 µl Dilute DNA to size Send to Genewiz 5 µl

12. Day 4: Machery-Nagel PCR Cleanup Protocol 1. Add all of the PCR content into 100 µl of Buffer NT 2. Load the sample into the MN column inside of a 2.0 ml microfuge tube 3. Centrifuge column for 1 minute at 11,000 Xg 4. Discard flow-through 5. Add 700 µl of Buffer NT3 into column 6. Centrifuge column for 1 minute at 11,000 Xg 7. Discard flow-through 8.Centrifuge column for 2 minutes at 11,000 Xg 9. Place column in new sterile 1.5 microfuge tube 10. Add 25 µl of sterile water to column

13. Day 4: Continued 11. Let column sit at room temperature for 1 minute 12. Centrifuge column for 1 minute at 11,000 Xg (DNA is in tube) 13. Test DNA in a spectrophotometer to verify its presence 14. Dilute DNA to 2µg/ml size 15. Transfer 10 µl of sized DNA template to PCR tube 16. Add 5 µl of 5 µM primer rpsl_UP to tube 17. Send tube to Genewiz for sequencing

14. Day 5: Sequence Analysis • Do a Blastn search • Go to http://www.ncbi.nlm.nih.gov/blast/Blast.cgi • Click ‘nucleotide blast’ • Click ‘others (nr etc)’ radio button • Enter ‘W3110’ into ‘Organism’ field • Check box next to “E. coli W3110” • Paste sequence into ‘Enter accession number(s), gi(s), or FASTA sequence(s)’ • Click ‘Blast’ • Do a Blastx search • Go back to the BLAST page with sequence already pasted • Select blastx • Click ‘blast’

15. Day 5: Sequence Analysis 3. Do a Multalin search • Go to http://multalin.toulouse.inra.fr/multalin/multalin.html • Test nucleotide sequence against wild type nucleotide sequence • Test amino acid against wild type amino acid sequence • Run multalin with other sequences