Griffitts:Bacterial DNA preparation

Materials

• Proteinase K
• Lysozyme
• RNase A
• Isopropanol (at room temperature)
• Chloroform (at room temperature)
• 70% ethanol (at 4°C)
• T10E1
• 5 M NaCl
• Binding Buffer
• 10% CTAB
• QiaPrep Genomic-tips (1 tip per sample)
• 1.7-mL microcentrifuge tubes
• 15-mL Falcon tubes

Buffer preparation

Lysozyme (1 mL)

• 100 mg lysozyme (in –20°C freezer)
• 1 mL ddH₂O
• Vortex
• Divide into 100 μL aliquots
• Store at –20°C

50 mg/mL RNase A (200 μL)

• 10 mg RNase A (in –20°C freezer)
• 200 μL ddH₂O
• Vortex
• Divide into 20 μL aliquots
• Store at –20°C

5 M NaCl (75 mL)

• 50 mL dH₂O
• 21.9 g NaCl
• This may require some heating
• QS to volume with dH₂O
• Autoclave

10% CTAB (10 mL)

• 6 mL ddH₂O
• 1.4 mL 5 M NaCl
• 1 g hexadecyltrimethylammonium bromide (CTAB)
• Alternate vortexing and 10-minute incubations in the 60°C water bath until dissolved
• QS to volume with ddH₂O

Binding Buffer

(This is the Qiagen Equilibration Buffer without isopropanol)

• 1.4 mL 5 M NaCl
• 1 mL 0.5 M MOPS
• 150 μL 10% Triton X-100
• 7.45 mL ddH₂O

Procedure

NOTE: Do NOT use pipetting for resuspension—it can cause shearing of the genomic DNA.

Lysis

1. Prepare all buffers and stock solutions beforehand and label all tubes
   • All buffers (except the Binding Buffer and the 70% ethanol) should be equilibrated to room temperature
   • Pre-warm the Binding Buffer to 50°C to increase yields
   • 70% ethanol should be cold (4°C)
2. Grow S. meliloti cultures in 4-mL LB overnight to saturation
3. Centrifuge two 1.5-mL aliquots of each culture at 13,200 rpm for 2 min. in 1.7-mL Eppendorf tubes
4. Discard supernatant
5. Thoroughly resuspend each pellet in 1 mL LB
6. Centrifuge at 13,200 rpm for 2 minutes
7. Discard supernatant
8. Resuspend each pellet in 735 μL T10E1
9. Add 39 μL 10% SDS to each tube
10. Invert several times
11. Immediately add 5 μL Proteinase K to each tube
12. Vortex 5 seconds
13. Incubate on the rotator at 37°C for 60 minutes

• Optional: Remove 300 μL and save for an analytical gel (test aliquot 1 = nuclear lysate)

CTAB Genomic DNA Isolation

1. Add 125 μL 5 M NaCl to each tube
2. Vortex 10 seconds
3. Add 100 μL 10% CTAB to each tube
   • Make sure the CTAB hasn't crashed out of solution; if it has, alternate vortexing and 10-minute incubations in the 60°C water bath until it is dissolved
   • At this point a precipitate should form
4. Vortex 10 seconds
5. Incubate on the 60°C for 15 minutes
6. Add 500 μL chloroform
7. Vortex 10 seconds and shake to make a milky emulsion
8. Centrifuge for 20 minutes at maximum speed
   • A well-packed interface should form
9. Remove ~800 μL of the aqueous layer (top layer) to a new tube
   • Do this slowly so that you don't suck up the debris at the interface
10. Add 2 μL RNase A
11. Vortex 5 seconds
12. Incubate at 37°C for 20 minutes

• Optional: Remove 8 μL and save for an analytical gel (test aliquot 2 = CTAB purification)

Genomic DNA Isolation

1. Suspend a QIAGEN Genomic-tip 20/G over a culture tube using a yellow tip holder
2. Equilibrate the tip with 2 mL Buffer QBT and allow it to empty by gravity flow
3. To the sample(s) from the lysis procedure add 800 μL Buffer B1
4. Add 350 μL Buffer B2
5. Vortex for 10 seconds
6. Add each sample to a tip and allow it to enter the resin by gravity flow
   • You may have to apply positive pressure with a syringe (4–10 drops/minute)
7. Remove 300 μL and save for an analytical gel (test aliquot 3 = flow-through fraction)
8. Move the tip and tip holder to a new culture tube
9. Wash three times with 1 mL of Buffer QC and allow it to move through by gravity flow
   • You may have to apply positive pressure with a syringe (4–10 drops/minute)
10. Remove 1200 μL and save for an analytical gel (test aliquot 4 = wash fraction)
11. Move the tip and tip holder to a new culture tube
12. Elute twice with 1 mL of 50°C Buffer QF and allow it to move through by gravity flow
    • You may have to apply positive pressure with a syringe (4–10 drops/minute)
13. Precipitate the DNA with 7 volumes of isopropanol
    • At this point and on, you should include your test aliquots
14. Vortex 10 seconds
15. Centrifuge at maximum speed for at least 15 minutes at 4°C
16. Carefully discard the supernatant
17. Wash with 1 mL cold 70% ethanol
18. Vortex 10 seconds
19. Centrifuge at maximum speed for 10 minutes at 4°C
20. Carefully discard the supernatant
21. Air dry for 10 minutes
22. Resuspend the DNA pellet in 50 μL of TE buffer
    • Drizzle the TE buffer twice down the back of the tube
    • The final concentration should be at least 5 ng in no more than 100 μL
23. Dissolve the DNA on a shaker for at least two hours (preferably overnight) at room temperature at 250 rpm
24. Analyze by gel electrophoresis on a 1% gel for 30 minutes at 120 V
    • For test aliquots, add 2 μL 8X DNA loading dye to 2 μL DNA and load 4 μL
    • For final DNA samples, perform a dilution series using 6 μL 8X DNA loading dye to 3 μL DNA (or 3 μL of the previous dilution) and load 4 μL

Adapted from QIAGEN Genomic DNA handbook and various CTAB procedures:

DOE JGI

Current protocols in Molecular Biology (.pdf)

The Nucleic Acid Protocols Handbook

Protocols for Nucleic Acid Analysis by Nonradioactive Probes

Sharon Long lab protocols.