DNA Plasmid Prep

In 1988 Kraft et al came up with and published a method for extracting DNA from E. coli using an alkaline lysis.

Since then the protocol has been modified (sometimes by companies who make kits for DNA extractions and sell them for lots of money to make the procedure easier).  I’m repeating one of those modifications here because being able to extract DNA from bacteria is one of the most common and useful ways of growing DNA.

Eventually it’s my hope to have a plasmid exchange at an upcoming site and this will be the most common way that exchange system will take advantage of growing DNAs.

I love nature.  Animals breed, plants grow and bear fruit, and bacteria replicate DNA.  All for nothing but some nutrients.

The first step is to transform bacteria with whatever DNA you want to grow.  DH5-alpha is a common choice for bacterial transformations and DNA preps.  It’s also possible to make your own E. coli chemically competent by adding some salts and things.  We’ll save that one for another time.

Supplies necessary for a DNA extraction are:

• culture tubes
• LB with selection agent
• Buffer P1* (premade)
• Buffer P2* (premade)
• Buffer P3* (premade)
• 1.5ml eppendorf tubes
• centrifuge
• a bucket of ice
• pipet tips or toothpicks
• 95% ethanol
• 70% ethanol
• Buffer TE

Once your bacteria is transformed and growing happily on agarose plates, grow a starter culture by picking a colony and innoculating 3 to 5mls of LB including your selecting agent (the two most common antibiotics for selecting bacteria are ampicillin and kanamycin).  When picking colonies I like to use pipet tips since they’re sterile and I can eject it from the pipetman straight into the tube so the tip doesn’t hit the side.  I find this much easier than using something like toothpicks.

Let the starter culture grow until it looks how you want it to (usually for me this means if I start the culture in the morning I do the prep in the late afternoon – 6 hours later or so).

1. Centrifuge the culture down.  I centrifuge it by separating it into 1.5ml eppendorf tubes, spinning them down for a couple minutes, then adding the pellets all into one tube and spinning down again.
2. Remove all the supernatant and resuspend the pellet in 100ul of Buffer P1.*  Let it sit for 5 minutes at room temperature.
3. Add 200ul of Buffer P2* and invert the tube a few times (gently).  Let it sit for another 5 minutes but this time on ice.
4. Add 150ul of Buffer P3* and vortex briefly or mix thoroughly by inverting.  Let it sit on ice for another 5 minutes.
5. Centrifuge for 10 minutes at 4 degrees (in the fridge or in a cold room).
6. Put the supernatant in a new tube and centrifuge again, same as before, 10 minutes in the fridge.
7. Decant the supernatant again into a new tube and add 1ml of 95% ethanol to the new tube.  Mix by inversion.
8. Put the tube on ice for 15 minutes and spin down again for 10 minutes at room temperature.  This pellets the DNA to the bottom.
9. Remove and discard the supernatant.
10. Add 1ml of 70% ethanol and centrifuge for 1-2 minutes.
11. Completely remove the supernatant and let the pellet dry.
12. Once it’s dry, resuspend the DNA in 20ul of TE.

And wallah!  DNA in a tube.

*Buffer P1:

• 50mM glucose
• 25mM Tris (pH 8.0)
• 10mM EDTA

*Buffer P2:

• 0.2N NaOH (or 0.2M – for NaOH it’s the same thing)
• 1% SDS (weight/volume [1g/100ml])

*Buffer P3:

• 60ml 5M potassium acetate
• 11.5ml acetic acid
• 28.5ml ddH2O