Griffitts:Bacterial DNA preparation

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Contents

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)

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.
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