Purification of bacterial genomic DNA

 A Summary of DNA Purification

November 13th, 2021

Overview

The process of DNA purification is one with many steps, and its uses are numerous, one of which is carrying out PCR reactions. During this week's lab, DNA is being purified and analyzed for four E. coli strains, two possibly missing the ProY gene (proY::kan) and two missing the BrnQ gene (brnQ::kan). These four strains were derived from last week's transduction procedure, and they were labeled GCC 104 and 108 for the delta ProY strain and GCC 201 and 210 for the delta BrnQ strain. The four transductants were grown in Dr. Deutch's lab at home in LB broth using colonies grown previously on Petri dishes. The purpose of this procedure is to isolate the genomic DNA from these four transductants and use it to perform a PCR reaction to confirm that last week's transduction of E. coli CDC11-1 with the JW5055 (delta ProY) and JW0391 (delta BrnQ) strains worked successfully. By the end of the procedure, we end up with pure DNA from the four chosen transductant strains (GCC 104, GCC 108, GCC 201, GCC 210). 

Procedure

For this procedure, a protocol for the DNeasy UltraClean Microbial Kit is being used. The steps of DNA purification can be summarized in the following bullet points:

• About 1.8 ml of each of the four transductant strains' cultures is added to a Microcentrifuge Tube and centrifuged for 1 minute to collect a pellet of cells without the supernatant. Repeat for the remaining 1 ml of broth culture. 
• These cells are then resuspended in 300 microliters of PowerBead Solution containing a buffer and salts to help homogenize the cells. 
• 50 microliters of Solution SL is added to the mixture which was transferred to a PowerBead Tube and vortexed for 10 minutes. Cell lysis takes place during this step. A centrifugation step for 1 minute follows to get rid of cell debris on the bottom of the tube. 
• After transferring the resulting supernatant to another Microcentrifuge Tube, 100 microliters of Solution IRS is added and the mixture is then incubated at 4 degree Celsius for 5 minutes followed by a centrifugation step. This ensures that non-desired cell debris and proteins precipitate to the bottom of the tube. The supernatant is then transferred to a clean Microcentrifuge Tube without disturbing the pellet of left over cell materials.  
• 900 microliters of Solution SB is then added to help the existing DNA bind to the MB spin Column membrane to which the resulting mixture is then transferred. This is done in 700 microliter batches to fit the spin column. A centrifugation step for 1 minute follows each transfer. DNA is now bound to the membrane of the spin column. 
• 300 microliters of Solution CB is added to the MB Spin Column to wash the DNA that's bound to the membrane from residue salts. The flow-through of the wash is discarded followed by centrifugation. 
• The MB Spin Column is transferred to a clean Microcentrifuge Tube and 50 microliters of Solution EB is added to the center of the membrane of the spin column. This releases the bound DNA into the Microcentrifuge Tube.
• DNA is now ready for different applications. It is then stored as a frozen stock solution. 

* The steps on the left side of the image are the ones used in this protocol. 

Conclusion

As shown above, the assay of purifying DNA is a complex process with many chances of error, so the resulting amount of DNA depends on the amount of bacterial cells used in the first step. DNA is an organic compound that needs to be handled carefully, thus numerous buffer solutions were used to guide the behavior of this compound. For example, the highly concentrated salts Solution SB ensures that DNA binds to the membrane silica of the MB Spin Column while Solution EB contains no salts and is slightly basic (PH 8) to unbind the DNA from the membrane and release it into the collection tube. Now, each of the four new strains (GCC 104, GCC 108, GCC 201, GCC 210) grown previously, two are delta ProY and two delta BrnQ, have a designated DNA sample for further analysis. One application for this purified DNA includes checking the concentration and purity of the samples using a NanoDrop. Another application includes performing a PCR reaction which will further confirm the genotypes of the above mentioned strains. All in all, DNA purification is an important stepping stone for many molecular applications, and the process has countable steps to minimize error during the production of a highly pure DNA solution. 

References

Deutch, C.E. (November 2021) Purification of genomic DNA for PCR reactions. Put Project at GCC.