Team:Hamilton McMaster/blog/2015-09-14
Miniprep! The E. coli Go to a Happier Place...
Welcome back everyone! As always, new week, new procedure. Today we explore how to use Miniprep kits to isolate the transformed plasmid from our overnight cultures (explained in last week’s post). There are many types of prep kits that can be used depending on the size of your culture (and therefore expected DNA yield), ranging from Miniprep, Midiprep, Maxiprep, Megaprep, and Gigaprep. Since we are only expecting a small yield of plasmid DNA, we use the Miniprep kit.
Miniprep kits can be ordered from many different companies, and they all have their own protocols tailored for their specific materials. The following is just a general procedure; any details may only apply to the kit we used so please refer to your kit carefully before trying yourself! Rather than teach you exactly how to perform a Miniprep, I hope that by the end of this post you understand a little bit of what’s going on while you perform a Miniprep. Now onto the procedure!
If you are opening a new kit, check all the solutions to see if any of them have special storage requirements. You may also have to add ethanol to some of the solutions. When you are ready, micropipette your bacterial culture into 2mL sample tubes. The amount of sample tubes used depends on how much yield of DNA you want; we usually use two sample tubes per overnight culture (note that not all of the culture is used!). Centrifuge your sample tube until all the bacteria pellets at the bottom, then discard the supernatent and fill the tube again using your overnight culture.
Almost balanced…
Yes! Balanced!
…
Next, add the suspension solution (included in the kit) to the sample tubes, and vortex them to mix. Lyse the cells using the solution provided. For example, if the lysis solution is basic there may also be a neutralization solution to be added, which is acidic. As for the hundreds of thousands of E. coli, well, let’s just say they go to a better place…
Centrifuge the tubes, and the cell debris will pellet at the bottom. At this point, the plasmids we want to harvest are in the solution. Using the appropriate Micropipette, transfer the lysate into a spin column-collection tube. They are provided with the kit, and an example is provided in the picture below.
The blue portion is the spin column, and is detachable from the clear collection tube. The spin column has a thin mesh-like material which will collect our plasmid, while everything else gathers at the bottom. Centrifuge the spin column-collection tubes so that this occurs, and discard the flow-through in the collection tube. Use the wash solution provided in the kit to refill the spin column, and centrifuge again to discard flow through (again). This step may be repeated several times with the wash solution, depending on your protocol.
Discard the flow through one last time, then centrifuge the tube without any solution to ensure that the spin column is dry. Transfer the spin column to a new collection tube, for example a sterile 1.5 mL sample tube, and discard the old collection tube. The new sample tube is what will hold your yield plasmid DNA by the end of this procedure, so make sure to label it properly before using it!
Add the elution buffer provided in the kit using the appropriate micropipette, and centrifuge (yes, again). This time, the buffer will bind to the filter in the spin column, displacing all the plasmid filtered out so far so that they flow into the sample tube and become eluted DNA. Discard the spin column, and store the DNA normally at -20°C. It is ready to be used in other synbio procedures.
