pET15b-based induction (pHB3-HIS6)
For the TAs:
Night before class does induction:
1. Pick a fresh, single colony from a L-Amp plate incubated overnight about 16 hours at 37 degrees. Inoculate into 2 ml L-Amp liquid in culture tubes used for minipreps and incubate at 37 degrees at 250 rpm in shaker in 510 lab for from 4.25 to 4.5 hours (no more, no less). Put the tube in the refrigerator for use the next day.
2. Prepare one sterile, 250 ml glass flask per group. Add 19.5 ml L-KAN (40 ug/ml) and store at room temp overnight.
3. Have some L-KAN on hand.
4. Prepare 60 ul aliquots of my 420mM IPTG stocks, one per group. Prepare 190 ul aliquots of 4X SDS-PAGE sample buffer, one per group. Both should stay on ice.
NOTE: Try to remember to have each student label three 1.5 ml tubes for sample collection in this lab. Each student will sample the culture and prepare samples for loading the gel (this is not clear from the lab protocol write up).
Late morning the day of the lab:
1. About 3 hours before the start of class, pellet cultures from previous day and re-suspend in 1 ml L-AMP.
2. Add 0.5 ml cells to 19.5 ml L-Amp in 250 ml flask. Incubate at 37 degrees, 250 rpm, in my water bath incubator in the BIO 510 lab. Incubate for 2.25 to 2.5 hours such that the at 2.5 hours of incubation, the time is 2:15 PM.
3. The students will sample before inducing, and then again before leaving. You should collect 1 ml samples from each group’s flask at 7 PM and then discard the cultures.
NOTE: Prepare 7 or 8 percent SDS-PAGE gels, one gel per group. They will blot them and we’ll probe the blots with Ponseau S, get photos, and then put them in blocking solution overnight. They’ll probe them the next day.
Probing conditions and solutions recipes are on the way.
Student Protocol
1. When you get to lab, there will be a 20 ml culture of your pAS4-16 + pHB3-HIS6 clone ready for you to begin. Remove a 0.6 ml, uninduced sample, add it to you “uninduced” 1.5 ml tube. Add 50 ul of IPTG to the culture to induce expression of nimA, and place the culture back in the incubator. Do this step quickly but don’t rush.
2. Pellet the cells in your 0.6 ml sample with a 20 second spin, remove all the medium by pouring or pipetting, and re-suspend the cells in 1 ml TE. Pellet the cells, re-suspend in 60 ul PBS, and store the sample at – 20 degrees.
3. At 4:30-ish, take another 1 ml “induced” sample, add it to your labeled tube, and treat as in step 2 above. The TAs will take a final sample at 7 PM.
4. Thaw your culture samples from the previous lab. Add 60 ul of 4X SDS-PAGE sample buffer to each tube. Use the 1ml syringe and 27 gauge needle provided to mix the samples and reduce their viscosity. Do the uninduced first, then induced #1, and induced #2. You will probably generate lots of foam doing this. Pellet the samples for 1 minute to reduce the foam back to a liquid. Stop when you have reduced the viscosity of the solution such that you can pipet it up and down with your pipetman.
5. Heat the samples at 85 degress for 3 minutes before loading on a SDS-PAGE gel. You will be provided with two MW markers. 1 = pre-stained markers. 2 = 6HIS-tagged markers. The information on these markers will be on the web site.
6. Load 40 ul of each sample per lane and all the volume of each marker. You should have 5 lanes per group: Pre-stained MW markers; Uninduced; Induced #1; Induced #2; 6HIS-tagged MW markers.
7. Run the gels and set up a western transfer as instructed by TAs. The blots will be blocked by TAs and provided to you for probing in the next lab period.