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 tube (5 per sample)
• 15-mL Falcon tubes (4 per sample)

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

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 in 100 μL TE buffer
23. Shake 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. Vortex the sample(s) from the lysis procedure at top speed for 10 seconds
4. 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)
5. Remove 300 μL and save for an analytical gel (test aliquot 2 = flow-through fraction)
6. Move the tip and tip holder to a new culture tube
7. Wash three times with 1 mL of Buffer QC and allow it to move through by gravity flow
8. Remove 1200 μL and save for an analytical gel (test aliquot 3 = wash fraction)
9. Move the tip and tip holder to a new culture tube
10. Elute twice with 1 mL of 50°C Buffer QF and allow it to move through by gravity flow
11. Remove 600 μL and save for an analytical gel (test aliquot 4 = eluate)
12. Precipitate the DNA with 1.4 mL of isopropanol and mix
13. Centrifuge at maximum speed for at least 15 minutes at 4°C
14. Carefully discard the supernatant
15. Wash with 1 mL cold 70% ethanol and vortex
16. Centrifuge at maximum speed for 10 minutes at 4°C
17. Carefully discard the supernatant
18. Air dry for 10 minutes
19. Resuspend the DNA pellet in 0.1–2 mL of TE buffer
    • The final concentration should be ???
20. Dissolve the DNA on a shaker overnight

Analysis

1. Add 7 volumes isopropanol to test aliquots 1–4 and your sample eluates
2. Rinse with 1 mL cold 70% ethanol
3. Drain well
4. Air dry for 10 minutes
5. Resuspend test aliquots in 20 μL of TE buffer and sample eluates in an appropriate amount of TE buffer
6. Add 5 μL loading dye
7. Run 10 μL samples on a 0.5% agarose gel at 120 V until the dye reaches the bottom of the gel
   • This takes approximately one hour

Adapted from QIAGEN Genomic DNA handbook.