Griffitts:Midiprep

Materials

 * Buffer LYS (in Nucleobond kit)
 * Buffer NEU (in Nucleobond kit)
 * Buffer EQU (in Nucleobond kit)
 * Buffer ELU (in Nucleobond kit), warmed to 60°C
 * 5 mg/mL RNase A
 * 100 mg/mL lysozyme
 * Isopropanol (at room temperature)
 * 70% ethanol (at 4°C)
 * T 3 E 0.3
 * Nucleobond columns (one per sample)
 * 40-mL centrifuge tubes, autoclaved (two per sample)
 * 1.7-mL microcentrifuge tubes (four per sample)
 * 50-mL Falcon tubes (one per sample)

5 mg/mL RNase A (100 μL)

 * 0.5 mg RNase A (in –20°C freezer)
 * 100 μL ddH2O
 * Vortex
 * Divide into 20 μL aliquots
 * Store at –20°C

100 mg/mL lysozyme (1 mL)

 * 100 mg lysozyme (in –20°C freezer)
 * 1 mL ddH2O
 * Vortex
 * Divide into 100 μL aliquots
 * Store at –20°C

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) * Draw a line at the 1-mL mark on the tubes used to collect the eluent
 * 3) * All buffers (except Buffer ELU and the 70% ethanol) should be equilibrated to room temperature
 * 4) * At a later step you will warm the Buffer ELU to 60°C to increase yields
 * 5) * 70% ethanol should be cold (4°C)
 * 6) Grow S. meliloti cultures in 100-mL LB overnight to saturation
 * 7) * Add 400 μL of the appropriate antibiotic(s) as needed
 * 8) Centrifuge two 40-mL aliquots of each culture at 10,000 rpm for 5 minutes at 4°C
 * 9) Discard supernatant
 * 10) Thoroughly resuspend each pellet in 5 mL RES Buffer (in fridge)
 * 11) Combine samples from the same original culture into the same tube
 * 12) * Be careful not to mix samples from different cultures!
 * 13) Add 100 μL 100 mg/mL lysozyme
 * 14) Vortex 5 seconds
 * 15) Incubate at room temperature for 10 minutes
 * 16) Add 10 mL Buffer LYS
 * 17) Invert 4 times
 * 18) Incubate at room temperature for 5 minutes
 * 19) Add 10 mL Buffer NEU
 * 20) Invert 8 times
 * 21) Centrifuge at 11,000 rpm for 5 minutes at 4°C

Midiprep Column

 * 1) Suspend a Nucleobond column over a 50-mL Falcon tube using a white tip holder
 * 2) * It may be necessary to put tape on the column so that it doesn't slide down too far
 * 3) Equilibrate the column with 12 mL Buffer EQU and allow it to empty by gravity flow
 * 4) Dump the supernatant from each sample into a column and allow it to pass through the filter by gravity flow
 * 5) Wash the column filter with 5 mL of Buffer EQU and allow it to move through by gravity flow
 * 6) * Apply to the sides of the filter, not the bottom
 * 7) Remove the column filter and discard
 * 8) Wash the column with 8 mL of Buffer EQU and allow it to move through by gravity flow
 * 9) Warm the Buffer ELU to 60°C (to increase yields) in one of the following ways:
 * 10) * Microwave for ~5 seconds
 * 11) * Set in the 60°C water bath for ~5 minutes
 * 12) Elute with 4.5 mL of 60°C Buffer ELU and allow it to move through by gravity flow
 * 13) Collect eluent in 4 microcentrifuge tubes (1 mL per tube)
 * 14) To each fraction add 1 μL of 5 mg/mL RNase A
 * 15) Invert twice
 * 16) Incubate at 37°C for 30 minutes
 * 17) Precipitate the DNA with 700 μL of isopropanol
 * 18) Incubate at –20°C for five minutes
 * 19) Centrifuge at maximum speed for 8 minutes
 * 20) Remove most of the isopropanol
 * 21) Centrifuge at maximum speed for 1 minute
 * 22) Remove the rest of the isopropanol with a P-200
 * 23) Drizzle 100 μL cold 75% ethanol down the back of each tube
 * 24) * Do not pipette up and down or vortex!
 * 25) Centrifuge at maximum speed for 2 minutes
 * 26) Remove all of the ethanol with a P-200
 * 27) Add 50 μL of T 3 E 0.3
 * 28) * Drizzle the TE buffer twice down the back of the tube
 * 29) * Do not pipette up and down or vortex!
 * 30) To dissolve the DNA, incubate at 60°C and flick once every minute for 15 minutes

Analysis
Adapted from the Nucleobond Xtra Midi handbook.
 * 1) Analyze by gel electrophoresis
 * 2) Run 5 μL on a 1% gel for 20 minutes at 120 V