Materials
Week 1 ( Isolating Mutants and Determining Mutant Phenotype):
1 Late Exponential phase culture of wild type E .Coli W3110, a K12 strain
2 Tryptose Blood Agar Base plates (TBAB)
In addition to these 2 TBAB’s, need 1 more TBAB plate for testing mutant clones after incubation.
1 TBAB plate containing 500 ug/ml Streptomycin
Plus 1 plate for testing mutant clones after incubation
4 Sterile tubes for the dilutions
1 Bottle of sterile diluting broth
1 1.5 Sterile microfuge tubes
Spreaders in ethanol, as well as Sterile Toothpicks for transferring clones
Week 2 (PCR reaction):
Sterile toothpicks to transfer mutant clones into water
100 μl of sterile water
25 μl the 2X PCR mix, which contains nucleotides, buffer, and Taq polymerase.
1 μl of 10 μM Primer 1 (rpsL_UP)
1 μl of 10 μM Primer 2 (rpsL_DOWN)
1 μl of suspended E. Coli
Week 3 (Machery-Nagel PCR cleanup):
100 μl of Buffer NT (pre-measured binding buffer)
2.0 mL microfuge tube
Additional 700 μl of Buffer NT3 (wash buffer)
A sterile 1.5 ml microfuge tube
25 μl of ultrapure water
Materials for Sequencing Reaction:
1 PCR tube
2 μl Premix (which contains polymerase, dNTP’s, and fluorescent ddNTP’s)
2 μl Sequencing Buffer
1 μl PCR product (from Week 2)
14 μl Water
Week 4: Data Analysis