Quantity Material Symbol

1 Late Exponential phase culture of wildtype E. coli W3110, a K12 strain

3 Tryptose Blood Agar Base plates (TBAB)

2 TBAB plates containing 500 ug/ml Streptomyocin

4 Sterile tubes for dilution

1 Bottle of sterile diluting broth

4 1.5 ml microfuge tube

Spreaders in ethanol

Sterile Toothpicks

25 µl 2X PCR

Quantity Material Symbol

1 10 µM Primer 1 (rpsL_UP)

1 10 µM Primer 1 (rpsL_DOWN)

147µl Sterile water

100µl Buffer NT

1 Machery-Nagel Column in a 2.0 ml microfuge tube

700 µl Buffer NT3, wash buffer

5 µl 5 µM primer (rpsl_UP)

1 PCR tubes

Day 1

3 ml 1 minute max speed

100 µl Re-suspend

10-6

100 µl

100 µl

1.

2.

3.

4.

Incubate at 37oC overnight

Incubate at 37oC overnight

Incubate at 37oC overnight

Day 1

1. Transfer 3 ml of original E. coli culture to a microfuge tube

2. Spin microfuge for 1 minute at max speed3. Decant supernatant 4. Re-suspend pellet in 100 µl sterile broth5. Transfer and spread content onto a TBAB + Strep plate

6. Spread 100 µl of 10-6 diluted original E. coli onto two TBAB plates

7. Incubate all three plates at 37oC 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 µl

1 µl

1. 95° for 5 minutes2. 30 cycles of

1. 95° for 1 minute2. 55° for 1 minute 3. 72° for 1 minute

3. 72° for 10 minutes

1.

2.

3.

4.

5.

100 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 minutes2. 30 cycles of

1. 95° for 1 minute2. 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

1.

2.

3.

4.

Day 4: Continued

=

sit at room temperature for 1 minute

Centrifuge column for 1 minute at 11,000 Xg

25 µl

5.

6.

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 tube3. Centrifuge column for 1 minute at 11,000 Xg 4. Discard flow-through5. Add 700 µl of Buffer NT3 into column6. 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 tube10. Add 25 µl of sterile water to column

Day 4: Continued

11. Let column sit at room temperature for 1 minute12. Centrifuge column for 1 minute at 11,000 Xg

(DNA is in tube) 13. Test DNA in a spectrophotometer to verify its

presence14. 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 tube17. Send tube to Genewiz for sequencing

Day 5: Sequence Analysis

1. Do a Blastn search 1. Go to http://www.ncbi.nlm.nih.gov/blast/Blast.cgi2. Click ‘nucleotide blast’3. Click ‘others (nr etc)’ radio button 4. Enter ‘W3110’ into ‘Organism’ field5. 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 search1. Go back to the BLAST page with sequence already pasted 2. Select blastx3. Click ‘blast’

Day 5: Sequence Analysis

3. Do a Multalin search1. Go to

http://multalin.toulouse.inra.fr/multalin/multalin.html2. Test nucleotide sequence against wild type nucleotide

sequence 3. Test amino acid against wild type amino acid sequence 4. Run multalin with other sequences