Inoculate, MiniPrep, DNA gel eletrophoresis

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Inoculate, Mini-Prep, DNA gel eletrophoresis
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Pipetman

• P20 2-20 ul
• P200 20-200 ul
• P1000 200-1000 ul

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Mini-Prep Protocol

• Start 2mL bacterial cultures containing
  plasmids of interest that have grown 12-18 hours
  at 37.
• Pour 1.4 mL of the culture into labeled 1.5 mL
  µfuge tubes. Place the borosilicate glass tubes
  with the remaining cultures at 4 they can be
  used to start additional culture for several
  weeks.
• Pulse down cells in microcentrifuge on high
  (14,000 rpm) for 30 seconds.
• Dump supernatant, suck off remaining culture, and
  resuspend cells in 100µL of ice cold Solution 1.
  It is important to resuspend all the cells and
  not leave large chunks of cells in the tube. To
  do this try rasping the microfuge tubes along the
  top of a microfuge rack or vortexing is ok.
• Add 200µL of freshly made Solution 2. Mix by
  gently inverting and rolling the tube to make
  sure all the bacterial slurry along the sides and
  cap are mixed with solution 2, incubate on ice
  until translucent ( 5 minutes- generally the
  first tube is done by the time you do 18-20
  samples).
• Add 150µL of ice cold Solution 3. Vortex
  immediately, incubate on ice 5 minutes.
• Microcentrifuge at 12,000-14,000 x g for 10
  minutes, remove supernatant to new
  microcentrifuge tubes. For cleanest preps
  avoid transferring any precipitate.
• Add 1mL of isopropanol to each tube (fill
  microcentrifuge tube w/ squirt bottle). Mix by
  inverting several times. Cold isopropanol or
  incubation on ice may increase DNA precipitation
  yields. May be stored in freezer at this stage
  if necessary.
• Microcentrifuge at 12,000-14,000 x g for 10
  minutes, carefully discard supernatant, and add
  1mL of 70 EtOH with a squirt bottle to wash
  pellet and gently mix by inverting. If pellet
  is disturbed recentrifuge for 5 minutes at
  12,000-14,000 x g.
• Pour supernatant into ethanol waste container,
  pulse down remaining EtOH in a microfuge at 5,000
  x g for 30 seconds, remove remaining EtOH with a
  pipettor, and allow remnants to evaporate on
  bench or in dessicator until pellet is just dry.
  Problems may be encountered trying to
  resuspend the pellet if allowed to overdry.

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• Reagents/Solutions
• Solution 1
• 50mM glucose
• 25mM TrisCl (pH 8.0)
• 10mM EDTA (pH 8.0) Filter-sterilize small batches
  of 100mL and store at 4 C. Glucose will
  caramelize if autoclaved.
• Solution 2
• 0.1M NaOH (freshly diluted from 10M stock)
• 1 SDS
• For 18 mini-preps (microcentrifuge head capacity)
• 3.76 mL dH2O
• 40uL 10M NaOH
• 200uL 20 SDS
• Solution 3
• 60mL 5M KAcetate (Final Concentration 3M)
• 11.5mL Glacial Acetic Acid (Final Concentration
  5M)
• 28.5mL dH2O

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pSilencer 3.1-H1 puro vector
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BamHIHindIII