Team:Hamilton McMaster/blog/2015-08-31
The Magical World of Transformations
Hi guys! Back to me again for this week’s blog entry (for any newcomers, I’m Fei Fei - nice to meet you!). Today I’ll be writing about a procedure I was very excited to learn and one that’s quite important in cloning. Transformation is the process through which we induce plasmid uptake in bacteria (in our case E. coli) in the hopes that the plasmid with our GOI (Gene of Interest) is introduced to that sample of bacteria. The survival mechanism we take advantage of is the tendency of E. coli to uptake surrounding plasmids and DNA when it is trying to survive and adapt to new environmental conditions. By quickly changing the temperature of our sample, we create extreme changes in the environment and cause the cells to uptake our plasmid. Now that we understand the purpose of transformations, let’s get to the procedure!
Remember: This blog only contains overviews of synthetic biology and microbiology lab procedures for educational purposes. The following values were used in our transformation recipe, and are not absolute values or ratios. If you plan on performing transformations, please refer to your official procedures beforehand!
First, thaw your competent cells and plasmid DNA samples on ice (just like we learned from Max last week! ^_^) for 30 minutes. With the appropriate micro pipette, add 100 uL of comp. cells and 10 uL of DNA into a 2 mL sample tube. One sample tube will be one sample of transformed bacteria by the end of this procedure. Remember to label your sample tubes, and keep track of the different plasmids you are using! Writing the date on the tube may also help avoid any future confusion when working with the same plasmid.
Now comes my favourite part of transformation! This step can be quite exciting and a bit stressful, since it is very time sensitive. No worries though, you’ve got this! Anxiety and rushing often cause mistakes, so just trust yourself and trust the timer :). First, set your heat shock device to 85°C so that will be at the right temperature when you use it. Place the sample tubes (with the comp cells and DNA) on ice for 20 minutes. In immediate succession, heat shock the samples in your device for 30 seconds at 85°C (use a clock or timer!) and then place them in an ice bath for 5 minutes. This is where the magic happens! You can’t see it, but your comp cells are reacting to the sudden heat and cooling, and taking in your plasmid - hence the name heat shock.
Next we move on to incubating colonies of our transformed culture. Unfortunately, this also means lots of waiting. Add 500 uL of SOC Medium to each of your sample tubes, and spin them in the incubator for 2 hours. This may seem like quite a lot of time, but it cannot be avoided. In the mean time, let’s prepare some LB agar plates to grow our cultures!
It’s always good to prepare a batch of plates beforehand, since it is more efficient and they can be stored for about a month. For the sake of learning though, let’s say we ran out and need to make some on the spot. Last week, Max talked about making LB agar solutions and selection through antibiotic resistance, and this is where these concepts apply. Make sure to use the LB agar solution with the antibiotic that corresponds to the resistance gene in your plasmid.
Prepare and Autoclave the solution to ensure sterility, and let it cool for about 5 minutes (until it is safe to touch but not cold enough to solidify). Label all the plates you will use, and light a Bunsen burner nearby to create an area of sterility. Slowly pour the LB agar onto each plate, keeping the lid partially over the plate with one hand for sterility, just until the agar fully covers the bottom (the plates don’t have to be thick, plus we’re on a budget so save the agar!).
After you pour a plate, you must not move it in any way until it is dry, otherwise it will not settle properly. Get rid of any bubbles in the agar using a toothpick. I found it convenient to pour plates from top rows to bottom rows, since that requires the least contortion on my part. The plates should be dry in 5-10 minutes, and then they can be stacked and stored upside down in the 4°C fridge. This prevents any condensation on the lid from dropping onto the agar. That’s it for pouring plates! Just in time, since our sample tubes have finished incubating. :)
Take out your samples, and centrifuge them for 1 minute, until the cells form a pellet at the bottom of the tubes. If you have an odd number of sample tubes, use an empty one to balance the centrifuge. We don’t want to damage expensive lab equipment!
Take out 500 uL of the supernatant (the remaining solution other than the pellet) with the appropriate micro pipette from each sample and discard it in the designated container. Vortex the remaining sample until the pellet is re-suspended in the solution. The vortex machine is basically the opposite of a centrifuge, and mixes a solution thoroughly instead of separating it by density.
Using a sterile smearing tool, smear your samples onto the LB agar plates you’ve prepared. Simply pour a drop of the sample onto the middle of the plate, and use the tool to gently smear the solution outwards in circular motions until you’ve covered the whole plate. Be careful not to break the agar!
Staged for demonstrative purposes - wear gloves as proper PPE!
Wait 5 minutes for the plates to dry, then place them into an incubator. You can stack the plates to save space, but remember to incubate them upside down! Check back next morning to see if your transformation was successful. Sometimes there are lots of colonies and sometimes you have to squint to find one the size of a speck of dust, but as long as there is something, rejoice! Even that speck is enough to be used in the next step. If nothing grows, no worries, cloning has never been an easy process. Just try, try again!
That’s it for transformations! Tune in next week to find out how Max turns even a speck of a culture into enough cells to work with by preparing overnight cultures, and how to make glycerol stocks for Cryogenic long-term storage. Have a great week!
Source: This knowledge was extracted from the brains of Hamza Q. and Sam S., who kindly gave explanations while we were waiting (there’s always something to wait for in microbiology). Thanks guys! :)