Pushing the Limits of the Electrophoresis Lab

For the second year now, we've been using Bio-Rad's forensic electrophoresis lab. My schedule is different than last year, so when I could prep for this lab had to be shifted. I poured the agarose gels on the Thursday of the week before we were going to do the lab and put them in the fridge with some buffer. I had planned to run the gels that Tuesday, so the gels would only be 5 days old.

I emailed the company about the feasibility of rehydrating the DNA samples and aliquoting them on Friday because I wasn't confident that I'd have enough time to do it Monday morning before we did the restriction digest. A Bio-Rad scientist confirmed that it would be fine.

Monday morning my students did the restriction enzyme digest. An hour later, we received the announcement that school would be closed on Tuesday because of how many students were out sick with the flu. On Tuesdays and Thursdays, we have 80 minutes for class, but on Wednesdays we only have 38 minutes. I knew 38 minutes wasn't long enough, so I called Bio-Rad and asked if our DNA would still be "runnable" if we waiting until Thursday. They confirmed that yes, it was possible and we could stick the samples in the freezer to be even safer.

Wouldn't you know it, we had a snow day on Thursday! Both Friday and Monday were only 38 minute periods for us, so I decided we'd just put it off until the next Tuesday. I figured even if it was a bust, I have pictures of successful gels and at least they would have the experience of filing the wells and watching the dye move through the gels as they were in the chambers.

The next Tuesday, 13 days after I had poured the gels and 9 days after we had done the restriction enzyme digest we ran our crime scene samples.

Although I followed Bio Rad's directions for running the gels in 20 minutes, I let them go for 45 minutes since the year before the bands hadn't moved very far. I think it's my power source. Anyway, by then my students had left for their next class, so I stained and destained their gels. I saw nothing right after. I figured the time lag was too long and I'd just show them pictures. I didn't toss the gels though since I wanted my students to see them anyway. I happened to take a look about 30 minutes later, and viola! There were the DNA bands. They weren't fabulous, but I think it has more to do with trying to run them rapidly. Next year I'll try the agarose concentrations for the longer running time and see how that goes.

Our Best Bacterial Transformation Lab Yet

Last year when we did our bacterial transformation lab, the results were lackluster at best. We had two transformed colonies on one of the LB/amp plates and one on a LB/amp/IPTG plate. We don’t have a digital water bath, and last year I struggled to keep the 42 degree water bath at 42 degrees on the hot plate. This is a heavy preparation lab to have poor results.

Since joining the National AP Biology Teachers group on Facebook, I had seen post after post of teachers raving about the transformation rates from the lab kit from Bio-Rad. So, when it was time to put in orders for this school year, we ordered the Bio-Rad Transformation kit through Wards.

The kit came in to school over the summer. I stopped by occasionally to check what orders were in. Then two weeks went by without me stopping in. When I finally came by to check, the kit was already sitting in the un-air conditioned storage room including the bag of bacteria, plasmids, ampicillin, and arabinose that says to refrigerate immediately. I took these materials out and got them into a refrigerator and started to worry that the kit was ruined even before we started. I hopped onto the Facebook group to see what people said about the kit and not refrigerating the items immediately.  I was surprised at how many teachers this happened to and was relieved to see that most of them had no adverse effects.

I had also shared with a colleague about how much I would like to have a digital water bath for this lab, but felt the cost was prohibitive. She suggested we try sous vide. I did some research and found an Anova Sous Vide Immersion Circulator for $109 on Amazon with good ratings and put it on our supply order. I brought in an old pot and put the circulator in. Within 10-15 minutes I had a water bath at exactly 42 degrees Celsius that remained at 42 degrees for as long as I wanted it there.

I did make a couple of other mistakes as I prepped for the lab. I started heating the LB agar in an (Ehrlemyer) flask on a hot plate. I stepped away to rehydrate the ampicillin and arabinose and heard a sizzling noise. I looked over to the hot plate to see agar erupting out of the flask. Fortunately the hot plate wasn’t plugged in behind the plate, but to the side of it, so I quickly unplugged it (and was glad I was wearing goggles, a lab apron, and closed toe shoes). Then I was panicked that I had lost too much agar to be able to pour all of the plates. I decided to scrape as much of the agar on the counter back into the flask and simmer it for 10 minutes to make sure it was sterilized. This time I stayed with it the whole time and there were no more volcanos.

My other mistake was that I rehydrated the ampicillin and arabinose with Luria broth instead of the transformation solution (calcium chloride). It wasn’t bad for these powders, but it meant I didn’t have enough broth left for my students to use in the lab. I doled it out carefully, but for the last group, I used left-over broth from the year before. (And I'm relieved to say they still had a successful transformation.)

I am happy to report that this year, we had some of our best transformation rates. I did have two groups with no transformation, but the other four had success. We certainly can’t blame the water bath!

Fortunately, I had enough agar to pour an extra LB/amp/ara plate for each lab group. We took the non-glowing transformed colonies from the LB/amp plates and swabbed them onto the spare plates. Students were able to see how the presence of arabinose really does switch on the jellyfish green protein gene when we looked at the plates the next day. This was one of our best years for this lab!