Griffitts:Bacterial DNA preparation

Materials

• Buffer QBT (in QIAGEN kit)
• Buffer QC (in QIAGEN kit)
• Buffer QF (in QIAGEN kit), warmed to 50°C
• Proteinase K
• 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
• 10% SDS

Buffer preparation

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 Buffer QF and the 70% ethanol) should be equilibrated to room temperature
   • Pre-warm the 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 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. Using a P200, 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)

Qiagen Genomic Column

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. Add 200 μL Binding Buffer to each 1.7-mL tube
4. Combine samples from the same original culture
   • Be careful not to mix samples from different cultures!
5. Add each sample to a tip and allow it to enter the resin by gravity flow
   • You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
   • Optional: Remove 300 μL and save for an analytical gel (test aliquot 3 = flow-through fraction)
6. Move the tip and tip holder to a new culture tube
7. Wash with 1 mL of Buffer QC and allow it to move through by gravity flow
   • You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
8. Wash again with 2 mL of Buffer QC and allow it to move through by gravity flow
   • You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
   • Optional: Remove 1.2 mL and save for an analytical gel (test aliquot 4 = wash fraction)
9. Move the tip and tip holder to a new culture tube
10. Elute with 2 mL of 50°C Buffer QF and allow it to move through by gravity flow
    • You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
11. Separate the eluate into 2 1.5-mL tubes (1-mL per tube)
12. Precipitate the DNA with 700 μL of isopropanol
    • At this point and on, you should include your test aliquots if you chose to collect them
13. Rock gently for 1 minute
14. Place tubes on ice for 15 minutes
    • The DNA precipitate should sink to the bottom of the tube
15. Centrifuge at maximum speed for 5 minutes
16. Remove most of the isopropanol
17. Centrifuge at maximum speed for 1 minute
18. Remove the rest of the isopropanol with a P-200
19. Add 100 μL cold 70% ethanol
    • Do not pipette up and down or vortex!
20. Centrifuge at maximum speed for 2 minutes
21. Remove all of the ethanol with a P-200
22. Add 60 μL of T10E1
    • Drizzle the TE buffer twice down the back of the tube
    • Do not pipette up and down or vortex!
23. To dissolve the DNA, incubate at 60°C and flick once every minute for 15 minutes
24. Combine DNA samples from the same original culture into one tube
    • Final volume should be 120 μL

Analysis

1. 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
   • The final concentration should be at least 5 ng in no more than 100 μL

Adapted from the 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.