Griffitts:Bacterial DNA preparation

Materials

• Buffer B1
• Buffer B2
• Buffer QBT (in QIAGEN kit)
• Buffer QC (in QIAGEN kit)
• Buffer QF (in QIAGEN kit)
• Qiagen Protease (in QIAGEN kit)
• Lysozyme
• RNase A
• Isopropanol (room temperature)
• 70% ethanol (cold)
• TE buffer
• QiaPrep Genomic-tips (1 tip per sample)
• 1.7-mL microcentrifuge tubes
• 15-mL Falcon tubes

Buffer preparation

Buffer B1 (40 mL)

• 32 mL dH₂O
• 745 mg Na₂EDTA·2H₂O
• 242 mg Tris
• 2 mL 10% Tween-20
• 2 mL 10% Triton X-100
• pH to 8.0 with HCl
• QS to 40 mL with dH₂O
• Store at 4°C

Buffer B2 (40 mL)

• 28 mL dH₂O
• 11.46 g guanidine HCl
• 8 mL 100% Tween-20
• QS to 40 mL with dH₂O

Protease

• Dissolve lyophilized Qiagen protease in 1.4 mL ddH₂O
• Store at 4°C

Lysozyme (1 mL)

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

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 Buffer QF and 70% ethanol should be equilibrated at room temperature
   • Pre-warm Buffer QF to 50°C to increase yields
   • 70% ethanol should be cold (4°C)
2. Grow S. meliloti cultures in 4-mL LB overnight to an OD₆₀₀ of 2.0
3. Pellet 2 mL of each culture at 10,000 rpm for 10 min. in 1.7-mL Eppendorf tubes
4. Discard supernatant
5. Resuspend each pellet in 1 mL Buffer B1
6. Add 4 μL RNase A to each tube
7. Add 20 μL Lysozyme to each tube
8. Add 45 μL Qiagen Protease to each tube
9. Vortex 5 seconds
10. Incubate at 37°C for 30 minutes
11. Add 350 μL Buffer B2
12. Invert five times
13. Incubate at 50°C for 30–60 minutes until sample becomes clear
14. If the sample is still cloudy, centrifuge at maximum speed for 10 minutes at 4°C
15. Remove 300 μL and save for an analytical gel (test aliquot 1 = nuclear lysate)

CTAB

1. Aliquot 700 μL samples into 1.7-mL Eppendorf tubes
2. Add 125 μL 5 M NaCl
3. Add 100 μL 10% CTAB
   • 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
4. Incubate at 50°C for 30 minutes
5. Add 700 μL chloroform
6. Vortex 10 seconds
7. Centrifuge for 10 minutes at maximum speed
8. Remove 500 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
   • Recombine your samples, but be sure not to mix them up!
9. Add 100 μL chloroform
10. Vortex 10 seconds
11. Centrifuge for 30 minutes at maximum speed
12. Remove 1 mL of the the aqueous layer (top layer) to a new tube
    • Do this slowly so that you don't suck up the debris at the interface
13. Add 700 μL isopropanol
14. Incubate for 30 minutes at –20°C
15. Centrifuge at maximum speed for 15 minutes at 4°C
16. Discard supernatant
17. Add 1 mL cold 70% ethanol
18. Vortex 10 seconds
19. Centrifuge at maximum speed for 15 minutes at 4°C
20. Discard supernatant
21. Air dry for 10 minutes
22. Resuspend the DNA pellet in 100 μL 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. 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.