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)
 * T 10 E 1
 * 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

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&mdash;it can cause shearing of the genomic DNA.

Lysis

 * 1) Prepare all buffers and stock solutions beforehand and label all tubes
 * 2) * All buffers (except Buffer QF and the 70% ethanol) should be equilibrated to room temperature
 * 3) * Pre-warm the Buffer QF to 50°C to increase yields
 * 4) * 70% ethanol should be cold (4°C)
 * 5) Grow S. meliloti cultures in 4-mL LB overnight to saturation
 * 6) Centrifuge two 1.5-mL aliquots of each culture at 13,200 rpm for 2 min. in 1.7-mL Eppendorf tubes
 * 7) Discard supernatant
 * 8) Thoroughly resuspend each pellet in 1 mL LB
 * 9) Centrifuge at 13,200 rpm for 2 minutes
 * 10) Discard supernatant
 * 11) Resuspend each pellet in 735 μL T 10 E 1
 * 12) Add 39 μL 10% SDS to each tube
 * 13) Invert several times
 * 14) Immediately add 5 μL Proteinase K to each tube
 * 15) Vortex 5 seconds
 * 16) 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
 * 4) * 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
 * 5) * At this point a precipitate should form
 * 6) Vortex 10 seconds
 * 7) Incubate on the 60°C for 15 minutes
 * 8) Add 500 μL chloroform
 * 9) Vortex 10 seconds and shake to make a milky emulsion
 * 10) Centrifuge for 20 minutes at maximum speed
 * 11) * A well-packed interface should form
 * 12) Using a P200, remove ~800 μL of the aqueous layer (top layer) to a new tube
 * 13) * Do this slowly so that you don't suck up the debris at the interface
 * 14) Add 2 μL RNase A
 * 15) Vortex 5 seconds
 * 16) 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
 * 5) * Be careful not to mix samples from different cultures!
 * 6) Add each sample to a tip and allow it to enter the resin by gravity flow
 * 7) * You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
 * 8) * Optional: Remove 300 μL and save for an analytical gel (test aliquot 3 = flow-through fraction)
 * 9) Move the tip and tip holder to a new culture tube
 * 10) Wash with 1 mL of Buffer QC and allow it to move through by gravity flow
 * 11) * You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
 * 12) Wash again with 2 mL of Buffer QC and allow it to move through by gravity flow
 * 13) * You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
 * 14) * Optional: Remove 1.2 mL and save for an analytical gel (test aliquot 4 = wash fraction)
 * 15) Move the tip and tip holder to a new culture tube
 * 16) Elute with 2 mL of 50°C Buffer QF and allow it to move through by gravity flow
 * 17) * You may have to apply positive pressure by mouth (1 drop every 5–10 seconds)
 * 18) Separate the eluate into 2 1.5-mL tubes (1-mL per tube)
 * 19) Precipitate the DNA with 700 μL of isopropanol
 * 20) * At this point and on, you should include your test aliquots if you chose to collect them
 * 21) Rock gently for 1 minute
 * 22) Place tubes on ice for 15 minutes
 * 23) * The DNA precipitate should sink to the bottom of the tube
 * 24) Centrifuge at maximum speed for 5 minutes
 * 25) Remove most of the isopropanol
 * 26) Centrifuge at maximum speed for 1 minute
 * 27) Remove the rest of the isopropanol with a P-200
 * 28) Add 100 μL cold 70% ethanol
 * 29) * Do not pipette up and down or vortex!
 * 30) Centrifuge at maximum speed for 2 minutes
 * 31) Remove all of the ethanol with a P-200
 * 32) Add 60 μL of T 10 E 1
 * 33) * Drizzle the TE buffer twice down the back of the tube
 * 34) * Do not pipette up and down or vortex!
 * 35) To dissolve the DNA, incubate at 60°C and flick once every minute for 15 minutes
 * 36) Combine DNA samples from the same original culture into one tube
 * 37) * Final volume should be 120 μL

Analysis

 * 1) Analyze by gel electrophoresis on a 1% gel for 30 minutes at 120 V
 * 2) * For test aliquots, add 2 μL 8X DNA loading dye to 2 μL DNA and load 4 μL
 * 3) * 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
 * 4) * 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.''