MINIPREP

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MINIPREP RESTRICTION ENZYMES

• WAKSMAN STUDENT SCHOLARS PROGRAM
• MARTY EDELBERG

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WHAT IS A MINIPREP?

• A plasmid DNA extraction from bacteria used to
  purify plasmid DNA.
• Miniprep is used when the starting E. coli
  culture volume is 15 ml of LB broth and the
  expected DNA yield is 2030 µg.

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MINIPREP EXTRACTS PLASMID
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WHY MINIPREP?

• Need to purify to be able to sequence
• Need to purify to be able to clone
• Need to purify to be able to digest and run on a
  gel (size insert)
• Need to purify to transform

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MINIPREP STEPS

• Start with overnight culture of E. coli
  containing plasmid with DNA insert of interest
• Tap the culture tube to resuspend cells and pour
  into 1.5 ml microfuge tube.

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MINIPREP STEPS-continued

• Spin microfuge in microcentrifuge for 1 minute at
  14,000 RPM
• Purpose-to form a pellet (separate cells from
  growth medium and concentrate cells)

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MINIPREP STEPS-continued

• Remove supernatant by pouring or pipetting

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MINIPREP STEPS-continued

• Resuspend the bacterial pellet by adding 200 ul
  of solution I
• Can vortex or pipett up and down to resuspend
• Pellet should be completely dispersed.

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PURPOSE OF SOLUTION I

• Glucose helps maintain osmolarity Tris is used
  to buffer pH of suspension
• EDTA chelates divalent cations (ions with a 2
  charge)
• Chelating Mg destabalizes the bacterial cell
  membrane and inhibits the action of DNAses that
  would destroy DNA
• Rnase destroys the large quantity of RNA in a
  cell.

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MINIPREP STEPS-continued

• Add 200 ul of solution II
• Mix gently by inverting 10 times
• Note a viscous bacterial lysate
• Aggressive mixing may shear chromosomal DNA into
  small fragments and contaminate prep

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PURPOSE OF SOLUTION II

• NaOH-Loosens cell wall and releases DNA,
  Denatures chromosomal DNA through linearization
  and separation (does not affect plasmid DNA)
• SDS-creates holes in cell membrane and denatures
  proteins
• Viscosity due to denatured chromosomal DNA

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MINIPREP STEPS-continued

• Add 400 ul of solution III
• Mix gently by inverting 5 times
• Note a white precipitate
• Aggressive shaking may break chromosomal DNA and
  contaminate prep

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PURPOSE OF SOLUTION III

• Sodium acetate-neutralizes NaOH
• Chromosomal DNA tries to renature at neutral pH
  but inefficient because completely separated due
  to its linear nature
• Salt ions-aggregate protein SDS complex causing
  them to precipitate.
• Chromosomal DNA gets trapped in precipitate
  before it can renature.
• Plasmids able to renature and remain soluble

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MINIPREP STEPS-continued

• Centrifuge for five minutes at full speed
• A white precipitate will form on the bottom and
  side of the tube
• Plasmid DNA remains in supernatant
• Chromosomal DNA and proteins in precipitate

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MINIPREP STEPS-continued

• Pour the supernatant into an appropriately
  labelled spin column that has been inserted into
  a collection tube
• Avoid adding the white precipitate
• Incubate at room temperature for 1 minute
• Microcentrifuge for I minute at full speed

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MINIPREP STEPS-continued

• Pour off the flow through from the collection tube

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MINIPREP STEPS-continued

• Add 400 ul of wash buffer to spin column
• Centrifuge at 1 minute at full speed
• Pour off the the flow through from the collection
  tube

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PURPOSE OF WASH BUFFER

• 80 ethanol to wash contaminates away from DNA
• Also contains Tris to buffer solution
• Also contains EDTA which chelates any metals that
  can be used by nucleases to degrade plasmid DNA
• Skipping this step will result in useless impure
  plasmid DNA

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MINIPREP STEPS-continued

• Centrifuge the spin column again for 1 minute at
  full speed
• Ensures removing any residual ethanol
• Place spin column in fresh microcentrifuge tube
  appropriately labelled

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MINIPREP STEPS-continued

• Add 60 ul of elution buffer-removes plasmid DNA
  from spin column
• Incubate at room temperature for 1 minute
• Centrifuge at full speed for I minute
• Throw away spin column and keep microfuge tube
  with liquid.
• Store in freezer.

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What tools do we use to cut DNA of interest and
join it to a plasmid or remove from plasmid?
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Restriction enzymes

• Proteins that cut DNA at defined sequences 4-8 bp
  long called restriction sites
• Cut phosphodiester bonds that link nucleotides
  together
• Cut in a precise and predictable manor, thus
  reproducible
• Restriction fragments-piece of cut DNA

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Where do restriction enzymes come from?
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Example-EcoR1 restriction enzyme
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How are restriction enzymes named?

• EcoRI from Escherichia coli
• BamHI from Bacillus amyloliqueraciens
• PvuI and PvuII are different enzymes from same
  strain.
• Genus-species-strain-order of discovery

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What restriction enzymes do we use in our
research?
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Sfi used to cut Duckweed DNA and plasmid for
joining
A.f. insert
Insert
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PvuII-cuts insert out of plasmid

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Serve as landmarks in plasmid to help find insert
when editing sequence

• Kpnl GGTACC
• SMA I CCCGGG
• ECORI GAATTC
• ScifiA GGCCATTACGGCC
• XBA I TCTAGA
• XHO I CTCGAG
• HIND III AAGCTT

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When we work with enzymes must create optimal
working environment

• Need buffer (pH, salt conc)
• Proper temperature-KEEP ON ICE
• Poor conditions may
• deactivate enzyme
• cause starr activity

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What is most important to remember?

• Always keep enzymes on ice (denaturation)
• Always use fresh tips
• Keep record in log book
• clone name
• date of digest

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DIGEST PROCEDURE

• Label I microfuge tube 5X Digest mix
• Label ____ tubes with clone name and digest (see
  how to label next slide)

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How do we label our clones?

• 13ME01.09
• 13PHHS
• MEInitial of person who made clone
• 01Number assigned to clone
• 09Year of project

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DIGEST PROCEDURE

• 1 Reaction mix
• dd H2O 7ul
• 10X buffer 2ul
• Miniprep DNA 10ul
• PvuII 1ul

• 5 reaction mix
• dd H2O 35ul
• 10X buffer 10ul
• Miniprep DNA
• PvuII 5ul

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DIGEST PROCEDURE

• Mix reaction mix by pipeting up and down
• Add 10 ul of reaction mix to each microfuge tube
  labeled with a clone name
• Add 10ul of the corresponding DNA to the
  corresponding labeled tube
• Mix each tube by tapping or in centrifuge at low
  for a few seconds
• Incubate for 1hour at 37C
• Add 2ul of 10X loading gel
• Store in freezer -20C