Streptococcus DNA Extraction - Genome Prep

Reagents Needed

• 50mM Tris-10mM EDTA, pH 8 Autoclaved.
  • Note: max concentration that will dissolve EDTA (without altering pH) is 0.1M. Making 10mL of the 50mM Tris Buffer Solution + 10mM of the EDTA will result in a 10% diluted Tris Buffer solution. We are working to troubleshoot this problem for the next time around.
  • If working with small volumes such as 10mL that need to be sterilized, an alternative to autoclaving is filter sterilization. Use either a 10 or 20mL syringe and 0.2 micro filter tip.
• 10 mg/ml lysozyme
• 10 mg/ml protease K
• 10 mg/ml RNAase A
• 10% SDS
• Tryptic soy broth blood plates
• Tryptic soy broth

If no 50mM Tris-10mM EDTA, pH 8 is available, you can make a stock solution:

1) Make a 1M Tris pH 8 solution

  A) Dissolve 54.92 g of Tris acid in 300 mL of Milliq water
  B) Dissolve 18.35 g of Tris base
  C) Add Milliq water until the total volume is 500 mL

2) Make a 0.5M EDTA pH 8 solution

  A) Dissolve 46.53 g of EDTA in 150 mL of Milliq water
  B) Add Milliq water until the total volume is 250 mL

3) Combine 35 mL 1M Tris, 14 mL 0.5M EDTA, and 651 mL water into one bottle.

Growing Up Strains

1. From glycerol stocks in -20°C freezer, get the strains you want to work with.
   • D39
   • Hermans 930
   • Pmen 4
2. Day 1:
   • Get Blood Plates (1 per strain), inoculation loops, tube holder. Bring everything into the hood.
   • Using an inoculation loop, streak from the glycerol stocks onto the entire plate. Change loops between strains.
   • Incubate plates upside down, O/N at 37°C
3. Day 2:
   • Using a sterile cotton swab, pick single colonies and streak them over half of a blood plate.
   • Incubate O/N at 37°C and 5% CO2.
4. Day 3:
   • For each strain, prepare 2 microcentrifuge tubes of 1ml tryptic soy broth each. Using a sterile cotton swab, transfer half of a blood plate of growth into a microcentrifuge tube with 1ml tryptic soy broth. Using the same cotton swab, swab this same half of the plate to get all bacteria off of it and place it in the second tube.
   • The salt is needed to precipitate the DNA in ethanol in later stages.
5. START extracting genomic DNA no later than 6 hours after you place cells in broth.
   • Ideally, start right away.

Lyse cells

1. Turn on small water bath, set for 37°C.
2. Spin down one tube per strain at full speed for 1 minute.
3. Remove supernatant. Just shake out the liquid, no need to pipette it out.
4. Pipet in 1ml of cells from the other TSB tube for the strain.
5. Spin down tube at full speed for 1 minute.
6. Remove as much medium as possible with a pipette, but do not disturb the pellet at the bottom of the tube or remove any cells.
7. Add 450uL of 50mM Tris-10mM EDTA, pH 8. Vortex to resuspend cells.
8. If having trouble resuspending cells, then let sit for 10-15 minutes before trying again.
9. Add 50uL 10 mg/ml lysozyme.
10. Incubate in 37°C water bath for 10 minutes.
11. Add 5uL 10 mg/ml protease K. [This is the same as proteinase K, if it is at a concentration of 20 mg/uL then use 2.5 uL]Vortex to mix.
12. Incubate in 37°C water bath for 30 minutes.
13. Add 5uL 10 mg/ml RNAase A and 50uL of 10% SDS
14. Invert tubes several times to mix.
15. Incubate in 37°C until the cultures are clear, this may take anywhere from 10 minutes to an hour.
16. Occasionally invert them to mix.
17. Spin them down at max speed for 10 minutes.
18. Remove the top 500uL (ONLY this volume) supernatant without disturbing the cell pellet.
19. Transfer this volume to a new micro-centrifuge tube.
20. If you disturb the pellet, re-spin. This is really important, don’t transfer any of the pellet.

Genomic Extraction of DNA

Use the DNA extraction kit (Zymogen Genomic DNA Clean-up and Concentration Kit)

1. Turn on the dry water bath and incubate a microcentrifuge tube of the DNA Elution Buffer or water (pH > 6.0) at 65°C
2. Add EtOH to the Wash Buffer as specified on the label. If you added EtOH, be sure to label such on the top cap and on the label. Then, be sure to close this bottle tightly each time you use it.
3. Obtain one 1.5mL microcentrifuge tube per strain and transfer 250uL of the lysed cell solution, so that there are two 1.5mL microcentrifuge tubes with 250ul each of lysed cells. Add 500uL of ChIP DNA Binding Buffer to each tube. Mix thoroughly through gentle inversion (not vortexing).
4. Do not be tempted to put this in one microcentrifuge tube; the contents will be at capacity and the lysed cell solution will splash out, potentially contaminating your gloves and/or the other DNA solutions.
5. Transfer one tube of this mixture to the Zymo-Spin IC-XL Column in a Collection Tube
6. Centrifuge (between 10,000-16,000 x g) for 30 seconds. Discard the flow through.
7. Transfer the other tube of this mixture to same column so that all 500uL if the lysed cell solution are added to the same column.
8. Centrifuge (between 10,000-16,000 x g) for 30 seconds. Discard the flow through.
9. Add 200uL DNA Wash Buffer to the column. Centrifuge for 1 minute. Repeat this wash step.
10. Add at 20uL DNA Elution Buffer or sterile water (pH > 6.0) from the 65°C dry bath directly to the column matrix and incubate at room temperature for five minutes. Transfer the column to a 1.5 mL microcentrifuge tube and centrifuge at max speed for 30 seconds to elute the DNA.
11. Add another 20ul DNA Elution Buffer or sterile water (pH > 6.0) from the 65°C dry bath directly to the column matrix and incubate at room temperature for five minutes. Transfer the column to a new 1.5mL microcentrifuge tube and centrifuge at max spped for 30 seconds to elute the DNA.
12. You’re now ready to use this DNA.

Quantify DNA

1. Quantify DNA from both final elution tubes with 1uL in the Superlab nanodrop (1st floor, east wing).
   • As long as there is more than 30ng/uL, we are good.