The first two weeks of the course will be a molecular biology "boot camp" in which the whole class will conduct a short project designed to help you master some basic techniques.
| SES # | ACTIVITIES | BOOT CAMP PROTOCOLS |
|---|---|---|
| 1 |
Start 150 ml culture of E. coli + pMQ64 in LB+30 ug/ml gentamicin Streak out PA14 from cryo-stock, put stock in own box in freezer | Day 1 (PDF) |
| 2 |
Maxi prep of E. coli+pMQ64 culture Set up digest of pMQ64 with BamHI Phosphotase cut vector, inactivate phosphotase, freeze vector at -20°C (vector will be gel purified at same time genomic DNA inserts are gel purified) Start overnight wt PA14, incubate in roller drum at 37°C | Day 2 (PDF) |
| 3 |
Isolation of genomic DNA from PA14 Pour 2 agarose gels | Day 3 (PDF) |
| 4 |
Get your DNA and it's concentrations from the teaching staff Set up your partial digest with BfuCI Load and run diagnostic gel Image 1st gel, analyze Set up upscaled digests Louad and run upscale gel; also run cut vector from Day 2 on this gel Image gel and cut out correct fragment size band from gel. Freeze at -20°C | Day 4 (PDF) |
| 5 |
Extraction of DNA from gel fragment Set up ligation: do 1:3, 1:1 and 3:1. Insert vector ratios, 2 no insert controls (one for each insert sample) & a no vector control (9 ligations) Transform ligations, do no DNA control here (cells only) (10 transformations) **Save the rest of ligation reaction in your freezer box** Spread x-gal on plates Plate 2x, 150 ul (20 plates/group) | Day 5 (PDF) |
| 6 |
Look at plates, calculate blue to white ratio for each ligation reaction and how many plates you need to pool to get good representation of PA14 genome in your plasmid prep. Pool colonies from appropriate number of plates and mini prep to purify plasmid mix. Freeze prep at -20°C. | Day 6 (PDF) |
| 7 |
Set up PCR on plasmid prep Pour gel for Day 8 Play with DNAStar | Days 7-8 (PDF) |
| 8 | Run gel to check results of PCR reaction and plasmid pool library |