QuantityMaterialSymbol 1Late Exponential phase culture of wildtype E. coli W3110, a K12 strain 3Tryptose Blood Agar Base plates (TBAB) 2TBAB plates containing 500 ug/ml Streptomyocin 4Sterile tubes for dilution 1Bottle of sterile diluting broth 41.5 ml microfuge tube Spreaders in ethanol Sterile Toothpicks 25 µl2X PCR

QuantityMaterialSymbol 110 µM Primer 1 (rpsL_UP) 110 µM Primer 1 (rpsL_DOWN) 147µlSterile water 100µlBuffer NT 1Machery-Nagel Column in a 2.0 ml microfuge tube 700 µlBuffer NT3, wash buffer 5 µl5 µM primer (rpsl_UP) 1PCR tubes

Day 1 3 ml 1 minute max speed 100 µl Re-suspend µl Incubate at 37 o C overnight Incubate at 37 o C overnight Incubate at 37 o C overnight

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 6. Spread 100 µl of diluted original E. coli onto two TBAB plates 7. Incubate all three plates at 37 o C overnight

Day 2 Incubate overnight 1.

Day 2: Determining if mutant is strep resistent, intermediate, or dependence 1. Count colonies on all three plates 2. With sterile toothpick transfer colonies from TBAB + Strep plates to a new TBAB + Strep Plate and then a TBAB plate 3. Incubate plates overnight

Day 3: PCR 22 µl 25 µl of 2X PCR mix 1 µl 1.95° for 5 minutes 2.30 cycles of 1.95° for 1 minute 2.55° for 1 minute 3.72° for 1 minute 3.72° for 10 minutes µl

Day 3: PCR 1. Add 22 µl of sterile water to 25 µl of 2X PCR mix (contains nucleotides, buffer, and Taq polymerase) 2. Add 1 µl of 10 µM Primer 1 (up) 3. Add 1 µl of 10 µM Primer 2 (down) 4. Add 1 µl of suspended E. coli 5. Place tube in thermocycler 1. 95° for 5 minutes cycles of 1.95° for 1 minute 2.55° for 1 minute 3.72° for 1 minute 3. 72° for 10 minutes

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

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

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

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

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

Day 5: Sequence Analysis 1.Do a Blastn search 1.Go to 2.Click ‘nucleotide blast’ 3.Click ‘others (nr etc)’ radio button 4.Enter ‘W3110’ into ‘Organism’ field 5.Check box next to “E. coli W3110” 6.Paste sequence into ‘Enter accession number(s), gi(s), or FASTA sequence(s)’ 7.Click ‘Blast’ 2.Do a Blastx search 1.Go back to the BLAST page with sequence already pasted 2.Select blastx 3.Click ‘blast’

Day 5: Sequence Analysis 3. Do a Multalin search 1.Go to Test nucleotide sequence against wild type nucleotide sequence 3.Test amino acid against wild type amino acid sequence 4.Run multalin with other sequences