Griffitts:Bacterial DNA preparation
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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
- 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)
- Grow S. meliloti cultures in 4-mL LB overnight to an OD600 of 2.0
- Pellet 1 mL of each culture at 10,000 rpm for 10 min. in a 1.7-mL Eppendorf tube
- Discard supernatant
- Resuspend each pellet in 1 mL Buffer B1
- Add 4 μL RNase A to each tube
- Add 20 μL Lysozyme to each tube
- Add 45 μL Qiagen Protease to each tube
- Incubate at 37°C for 30 minutes
- Add 35o μL Buffer B2
- Vortex briefly
- Incubate at 50°C for 60 minutes
- Centrifuge at maximum speed for 10 minutes
- Remove 300 μL and save for an analytical gel (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
- Remove 300 μL and save for an analytical gel (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 300 μL and save for an analytical gel (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 (aliquot 4 = eluate)
- Precipitate the DNA with 1.4 mL of isopropanol and mix
- Centrifuge at maximum rpms for at least 15 minutes at 4°C
- Carefully discard the supernatant
- Wash with 1 mL cold 70% ethanol and vortex
- Centrifuge at maximum rpms 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
- Dissolve the DNA overnight on a shaker overnight
Analysis
- Add 7 volumes isopropanol to aliquots 1–4
- Rinse with 70% ethanol
- Drain well
- Resuspend in 20 μL of TE buffer
- Add loading dye
- Run 10 μL samples on a 0.5% agarose gel until the dye reaches the bottom of the gel
Adapted from QIAGEN Genomic DNA handbook.