Aliquoting Guide for Lab 6
Aliquoting Guide for Lab 6
We have provided sufficient reagents for the number of students you indicated on the Materials Request form, including a bit extra for pipetting errors. All solutions should be aliquoted into the provided 1.5 mL microcentrifuge tubes, unless otherwise indicated.
See teacher instructions for Lab 6 on methods for growing the culture of transformed cells. We recommend doing this step with the commercially ligated/purified plasmid used in the short series (pARA-R). The sterile broth provided has been pre-calculated so there is enough for each group to get the 2mL required.
NOTE: You must grow the culture at least 24 hours to get good expression of the rfp before you can begin Lab 6.
Day 1. Lysing of cells from the overnight mFP expression.
| Label tube | Contents of tube | Aliquot | Actually used |
|---|---|---|---|
| EC | overnight E. coli culture | 1mL**** | 2mL |
| LyB | Lysis buffer | 160µL | 150µL |
| EB | Elution buffer | 160µL | 150µL |
****You will need to do this twice into the same tube, once before and once following centrifugation after the supernatant has been discarded.
Day 2. Purification of RFP using column chromatography
| Size of tube | Label tube | Contents of tube | Aliquot | Actually used |
|---|---|---|---|---|
| 15 mL | BB |
Binding buffer |
250µL | 200µL |
| 15 mL | CEB |
Column equilibration buffer |
3.5mL | 3mL |
| 15 mL | WB |
Wash buffer |
1.5mL | 1mL |
| 15 mL | EB |
Elution buffer |
2.5mL | 2mL |
Be certain that the last group of students flush the columns with 2 mL of equilibration buffer. Allow the equilibration buffer to drain but leave about 0.5cm of it above the resin bed. Be certain that all tubes are capped, that the extensions have been removed and small yellow caps replaced, and check to see that the stop cocks are in the off position before storing for the next school or class. Always store the columns UPRIGHT. Return the columns to the plastic, lidded box.