Incubated a fresh squid (from haymarket friday night) in a plastic bag for 2 days in a refrigerator. Does it glow?

answer: not yet. Smells, though.

Transferring it to a tupperware container cracked to provide some airflow.

Incubating with a cover on the benchtop.

The plan:

pipetted & spread 100 uL of 0.1 mM IPTG onto AMP plates, let dry.

Picked a J04450 colony and streaked it on two plates 30 min later. Looking for induction of the RFP in J04450 via IPTG repression of the LacI repressor.

24 hours later, no visible induction :(

Note, spilled ~3 mL of 1M IPTG after sterilizing it. MSDS had no clear clean-up information. Wiped up the spill w/ paper towel and put it w/ autoclave waste. IPTG is heat-sensitive. Washed the table 3x with water and then with bleach.

It would be excellent to have more operable “scram” instructions for situations like this (exactly what to do if a spill or exposure) before working with chemicals. Would like to develop these every time a new chemical is ordered, before it arrives at the lab. Not sure what the best way is to do.

Went over to the bosslab yesterday (16 May 2011) around 11 PM to check on the plates (18+ hrs of incubation in the “incubator”).

No signs of colonies. Transformation failed, or amp was too conc, or incubator was too hot, or…

SOC broth that was incubated was mildly turbid. The biobrick card used was not sterile, so the organisms were probably not transformed E. coli. Plated 100 uL of the SOC on LB agar plates anyway. We’ll see.

lab notes in evernote

The “incubator” we’re using at the bosslab is really an oven. Instead of temperature gradations on its control knob, it just has 5 numbers, “1-5”. With some trial and error we’ve got it set to a temperature between 28 C and 40 C.

But I wanted to see how responsive the temperature control system in the oven incubator is, so I set up a simple data-logger to record the temperatures inside it.

A Dallas Instruments temperature sensor (DS18B20) is hooked up to an arduino, which sends the temperature measurement once a second to a computer via “serial”. A processing sketch on the computer uploads the data to Pachube.

Here’re two dynamic graphs of the temperature data stream from pachube. (a dotted line indicates a data failure; probably because the computer was shut off, fell of the network, processing stalled… etc).

As you can see, the values oscillate on a roughly hourly cycle over a range of about 10 degrees C. I wonder how that affects the growth of E. coli… hmmm.

Goal: transformation of Invivogen lyocomp cells w/ BBa_J04450 on card (circa 2008).

• OWW Lab Notebook

• Photo set on Flickr

We’ll be back in 18 hours w/ photos of the results. Here’s what we did today:

Preflight

what’s our goal

dry-run through transformation protocol w/ BBa_J04450 on card using Invivogen lyocomp cells

• lyocomp 116 cells, e. coli k12 (?), non-pathogenic
• hot water bath; 42C, heat caution
• Ian: “The chemicals we’ll be using amount to waste water (E. coli), cheese whey (tryptone), Vegemite (yeast extract & NaCl), tofu firming salts (MgCl2 and MgSO4), jelly (agar) and antibiotics and the concentrations are fairly low, so no special dangers - i.e. don’t eat it. Cleanup can be city sewer, since all of these are things that are already found in waste water.”

risks to us

• trivial; just doing a dry run w/ the water baths
• conventional fire risk w/ propane burner for autoclave - use it outside on asphalt

risks to community

• fire hazard from propane burner

what waste are we gonna generate

• hot water
• combusted propane gas

Protocol

1. Thaw competent cells (Lyocomp E. coli) on ice.
2. During this time pre-chill sterile centrifuge tubes on ice.
3. Gently tap competent cell containers to mix
4. Aliquot bacterial suspension in pre-chilled tubes (100 µl per tube).
5. To the aliquots add 1 µl of plasmid (RFP PUC19).
6. Gently tap centrifuge tubes to mix
7. Incubate 30 min on ice.
8. During this time pre-heat water bath to 42°C (don’t use heating block).
9. Incubate samples at 42°C in the water bath for 60s. Do not shake.
10. Immediately chill samples on ice for 2 minutes.
11. Add 0.95 ml of room temperature SOC.
12. Plate 10 µl of bacterial suspension on antibiotic-containing agar plate. (filter sterilized Kanamycin100 ug/ml?).
13. Transfer some bacteria to another antibiotic-containing plate using a loop.
14. Incubate plates at 37°C for 18 h.

visual protocol

Earlier today I emailed all of the participants in the recent Reading Genes workshop. Here is what I said:

Thanks so much for participating this weekend in the diy-genotyping workshop. I personally had a great time working with all of you and I learned a wonderful amount preparing and running the event. It was a bit of an experiment, and if some or all of the results are negative, rest assured that I will figure out what went wrong and invite you back for a successful second try.

Today I’m purifying everyone’s PCR products for sequencing. I’m using an enzyme I bought from Affymetrix (the ExoSAP-IT kit) to do the purification. The sequencing machine wants 12 uL (micro-liters) of each person’s purified PCR Product. So, I’ll take 12 uL of unpurified PCR mix from each person’s tube, add it to a new tube, and also add 4.5 uL of the magical ExoSAP-IT mix.

Theoretically, the included enzyme will chew up any remaining non-PCR-product DNA and remove their phosphate backbone. The genomic DNA we copied, the primers, and the DNA nucleotide building blocks that we built the copy from (the As, Gs, Cs, and Ts) will be changed from being nucleoTIDES to nucleoSIDES. I can’t give the sequencer tubes with active ExoSAP-IT, since it would probably mess up the sequencing reactions (we’re using “Sanger sequencing”, which is like a specialized kind of PCR + gel electrophoreses), so I’ll denature the ExoSAP-IT after it does its work by heating the entire mix to 80 C for 15 minutes.

So that’s the plan. I’ll let you know how the sequencing goes.