40 Activity 1: Bacterial Lysis

1. Preheat two Eppendorf Thermal mixers with 1.5 ml blocks to 37°C and 55°C.

2. Use a sterile cotton swab to obtain bacterial colonies and resuspend in 300 µl of cold PBS or Tris buffer.

3.Centrifuge each microtube of the sample at the maximum speed setting (> 12,000 x g) for 1 minute.

a. Balance the centrifuge with the placement of microtubes. Use empty tubes if necessary.

b. After centrifuging, there should be a “pellet” of bacteria at the bottom of the tube.

c. Depending on the concentration of bacteria in the solution, this may take a few cycles of centrifuging, removing supernatant, and adding more sample to get a sufficient pellet.

4. Discard the supernatant. There are two methods for this depending on the strength of the pellet.

a. If the pellet is tightly packed (small clump at the bottom, little movement when swirled), you can simply dump the supernatant into the biohazard bin. The pellet will remain at the bottom. This method is commonly called “decanting the supernatant.”

b. If the pellet is not packed together at the bottom, first re-centrifuge up to two additional times. If it remains loose, remove the supernatant with a micropipette.  This method is commonly called “aspirating the supernatant.”

i. Start removing supernatant from the top of the meniscus and lower the tip as the meniscus lowers so as to not remove any of the pellet. A small amount of remaining supernatant is acceptable.

5. Resuspend the bacterial pellet in 300 µl of cold PBS or Tris buffer. Add 10 µl Lysozyme (50 mg/ml) solution and mix by vortexing for 1-2 seconds.

a. If the pellet remains at the bottom after vortexing, try flicking the tube. The cells will be lysed shortly, so you don’t have to be too gentle. Use a micropipette to pull the pellet off the bottom if it continues to stick to the bottom of the tube.

6. Add 300 µl HMW gDNA Tissue Lysis Buffer to the sample and mix by inverting 5-10 times.

7. Incubate at 37°C in a thermal mixer with agitation at 1400 rpm for 15 minutes, or until nearly clear (up to 20 minutes).

a. The samples may not become fully clear – that’s ok. If samples haven’t cleared up at all after incubation, ask the instructor for assistance.

8. Remove the tubes after finishing incubation, and set the thermal mixer to 56°C (or use one already set to this temperature).

9. Add 20 µl of Proteinase K and mix by inverting 10–20 times.

a. Keep the Proteinase K in or near the surface of the ice as much as possible; it is very temperature sensitive.

b. The Proteinase K is thick and will stick to the outside of the pipette tip. When pipetting, just barely break the surface so as to not end up with excess on the outside of the pipette tip. Do the same when depositing into the sample tube.

10. Incubate at 56°C for 30 minutes in a thermal mixer at 1400 rpm.

11. Add 10 µl of RNase A and mix by inverting 5–10 times. Incubate for 10 minutes at 56°C with agitation in a thermal mixer at 1400 rpm.

12. Add 300 µl of Protein Separation Solution. Mix by inverting gently for 1 minute.

13. Centrifuge for 10 minutes at maximum rpm.

a. The sample will separate into a large, clear upper phase (DNA) and a lower, clear phase (protein, usually on the bottom of the tube, but occasionally floating). There may also be a white precipitate at the bottom of the tube.

b. It may take longer than 10 minutes for complete phase separation to occur. Centrifuge for no more than a total of 25 minutes.

14. If working with multiple samples, prepare and label the plastics for the upcoming steps. Each sample will require:

a. Monarch Collection Tube II (not labeled)

b. 1 Monarch Bead Retainer inserted into the collection tube; this will be used to remove the wash buffer from the gDNA bound to the beads. (labeled)

c. 2 Monarch 2 ml Tubes; one for phase separation and one for elution. (labeled)

d. 1 1.5 ml microfuge tube; this will be used to collect the eluate. (labeled)

15. Using a 1000 µl wide-bore pipette tip, transfer the clear upper phase (containing the DNA) of each sample to a corresponding labeled Monarch 2 ml Tube. Typically, the transferred volume is ~800 ul. If less than 700 ul, ask your instructor about adjusting the volumes of solutions added in the next steps.

a. It’s important to remove as much of the upper phase as possible without removing the proteins collected at the bottom of the tube. If you’re having trouble pulling only the upper layer, try using a 200 ul pipette. A small amount (1-2 ul) of protein will not greatly affect your results.

b. If there isn’t a visible protein layer at the bottom of the tube, leave ~30 ul solution in the tube to be sure very little protein is transferred.