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

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 yeilds
   • 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 1 mL of each culture at 10,000 rpm for 10 min. in a 1.7-mL Eppendorf tube
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. Incubate at 37°C for 30 minutes
10. Add 350 μL Buffer B2
11. Vortex briefly
12. Incubate at 50°C for 60 minutes until sample becomes clear
13. If the sample is still cloudy, centrifuge at maximum speed for 10 minutes at 4°C
14. Remove 300 μL and save for an analytical gel (test aliquot 1 = nuclear lysate)

Genomic DNA Isolation

• Suspend a QIAGEN Genomic-tip 20/G over a culture tube using a yellow tip holder
• Equilibrate the tip with 2 mL Buffer QBT and allow it to empty by gravity flow
• Vortex the sample(s) from the lysis procedure at top speed for 10 seconds
• 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)
• Remove 300 μL and save for an analytical gel (test aliquot 2 = flow-through fraction)
• Move the tip and tip holder to a new culture tube
• Wash three times with 1 mL of Buffer QC and allow it to move through by gravity flow
• Remove 1200 μL and save for an analytical gel (test aliquot 3 = wash fraction)
• Move the tip and tip holder to a new culture tube
• Elute twice with 1 mL of 50°C Buffer QF and allow it to move through by gravity flow
• Remove 600 μL and save for an analytical gel (test aliquot 4 = eluate)
• Precipitate the DNA with 1.4 mL of isopropanol and mix
• Centrifuge at maximum speed for at least 15 minutes at 4°C
• Carefully discard the supernatant
• Wash with 1 mL cold 70% ethanol and vortex
• Centrifuge at maximum speed for 10 minutes at 4°C
• Carefully discard the supernatant
• Air dry for 10 minutes
• Resuspend the DNA pellet in 0.1–2 mL of TE buffer
  • The final concentration should be ???
• Dissolve the DNA on a shaker overnight

Analysis

• Add 7 volumes isopropanol to test aliquots 1–4 and your sample eluates
• Rinse with 1 mL cold 70% ethanol
• Drain well
• Air dry for 10 minutes
• Resuspend test aliquots in 20 μL of TE buffer and sample eluates in an appropriate amount of TE buffer
• Add 5 μL loading dye
• 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.