The format of this protocol for bacterial DNA extraction for the purpose of human genome editing is written in a step-wise fashion, with detailed explanations of the major steps in the process underneath that step.
Lab 6—Bacterial Chromosomal Extraction and Purification Protocol
1. Transfer bacterial cell culture (~1.5 mL grown in LB medium) to a 1.5 mL Eppendorf tube and centrifuge at 7500 rpm for 10 minutes.
a. Rationale: Bacterial are grown in LB medium, LB standing for lysogeny broth, a culture media primarily used to rapidly grow bacteria.
b. Centrifugation step is necessary to form a cell pellet at the bottom of the Eppendorf tube.
2. Aspirate the supernatant.
a. Removal of the remaining LB media so that we are only left with the bacterial cell pellet.
b. Note: be careful not to aspirate any of the cell pellets, as this will cause a high loss in the DNA yield.
3. Resuspend the cell pellet in 600 uL of a lysis buffer.
a. Lysis buffer recipe (10 mL): 9.34 TE buffer, 600 uL of 10% SDS, 60 uL proteinase K.
b. TE Buffer is a combination of Tris-Cl which is a pH buffer solution (prevent changes in pH of the solution) and EDTA which is a chelator of divalent cations (magnesium and calcium—ions that stabilize DNA structure).
c. SDS—sodium dodecyl sulfate, is a detergent that solubilizes the proteins/lipids that form the membrane of the cell.
d. Proteinase K—an enzyme that denatures various proteins found in the cell.
4. Incubate for 1 hour at 37 C.
a. Give ample time for the lysing process to occur.
5. Add phenol/chloroform at a 1 to 1 ratio. Mix by inverting the tube until no noticeable phase difference exists. DO NOT VORTEX TO MIX, this can damage the DNA.
a. Phenol is an acidic solvent which denatures the DNA, causing the DNA to partition to an organic phase.
b. Chloroform is an organic solvent that can then dissolve the denatured DNA.
6. Centrifuge at max speed (7,500 rpm should suffice) for 5 minutes (He, 2011).
a. This is to separate the various phases from each other. There should be a white layer at the phenol/chloroform interface. This is the protein layer.
7. Transfer the upper, aqueous layer to a new tube using a 1 mL pipettor.
8. Add an equal volume of chloroform to the aqueous layer and mix via inversion of the tube.
a. This will help remove the phenol.
9. Centrifuge again at max speed for 5 minutes.
a. Once again to help separate out the various layers.
10. Remove the aqueous layer.
a. This layer is not needed an can be properly discarded.
11. Add 2.5—3 mL of 200 proof ethanol (stored at -20 degrees C).
a. This step is to precipitate the DNA.
12. Mix the suspension gently.
a. Ensure ethanol is well mixed with the remainder of the solution.
13. Keep DNA/ethanol tube at -20 degrees C for a minimum of 30 minutes.
a. Allow for the precipitation reaction to occur.
b. Increase the time to increase the yield of the extraction.
14. Centrifuge the tube at max speed for 15 minutes. Make sure the centrifuge has had time to decrease the temperature to 4 degrees C.
a. This step is to form the DNA pellet. The pellet will look transparent when it is wet and will turn white when it becomes dry (Salva-Serra et al., 2018).
15. Aspirate the supernatant.
a. Remove the remaining ethanol-containing solution, leaving only the DNA pellet.
16. Wash the DNA pellet with 70% ethanol (stored at room temperature, the other 30% is deionized water).
a. Dilute/remove any remaining solution that was not aspirated previously.
17. Centrifuge tube at max speed for 2 minutes.
a. Help dilute any remaining solution that was not aspirated previously.
18. Aspirate the supernatant.
a. Remove the 70% ethanol solution, leaving the precipitated DNA pellet.
19. Air-dry the DNA pellet (for faster results this can be done in the 37 degrees C incubator.)
a. Evaporate any remaining ethanol solution.
20. Resuspend the DNA pellet in TE buffer.
a. You can add RNase proteins at this step to digest any RNA that is present in the solution. This can also be supplemented in the lysis buffer (step 3).
21. Test DNA on an agarose gel.
a. This step is to confirm that the procedure was successful, thereby increasing the reliability of testing in forensic science.
He, F. (2011). E. coli Genomic DNA Extraction. Bio-protocol Bio101: e97. DOI: 10.21769/BioProtoc.97.
Salva-Serra, F., Svensson-Stadler, L., Busquets, A., Jaen-Luchoro, D., Karlsson, R., Moore, E. R. B., & Gomila, M. (2018). A protocol for extraction and purification of high-quality and quantity bacterial DNA applicable for genome sequencing: a modified version of the Marmur procedure. Nature Protocol Exchange. doi:10.1038/protex.2018.084