Streptavidin purification of DNA fragments

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Contents

Biotin Primers

  • Make primers for PCR reactions with a 5' biotin modification
    • HPLC or PAGE purification of these primers is desirable to eliminate short primers and ones without a biotin tag
    • Virtually all oligo manufacturers can supply 5' biotin
    • An alternative is to make 5' amine primers and link biotin to the amine group


PCR Reaction

  • Normal PCR reaction conditions apply.
  • Approximately 1 pmol/μl biotinylated primer should be used.


  • 100 μl reaction
  • 93 μl PCR Supermix High Fidelity (Invitrogen)
  • 3 μl Suffix-FB biotinylated primer (30 pmol/μl)
  • 3 μl Prefix-RB biotinylated primer (30 pmol/μl)
  • 1 μl diluted plasmid backbone template DNA (1 ng/μl)


  • Cycle 30x
  • initial denature 95° 2 min
  • 30 cycles
    • 95° 15 sec
    • 55° 15 sec
    • 68° 3:30 min (possibly longer for very large backbones)
  • final extension 68° 20 min


Post PCR Cleanup

  • Elimination of PCR enzymes and dNTPs is required prior to enzymatic cutting
  • Add 1 μl Proteinase-K
  • digest at 50° for 1 hour
  • heat kill Proteinase K at 80° for 20 minutes
  • Add 5x (500 μl) Qiagen buffer PB, vortex
  • Spin in Qiagen column at 8000g 1 minute
  • Pour flow through back into the column, spin again
  • Discard flow through, add 500 μl buffer PB, spin again
  • Discard flow through, add 750 μl wash PE, spin again
  • Discard flow through, spin again at 12000g, 2 minutes to dry
  • Transfer column to a clean 1.7 ml tube, add 30 μl EB, spin at 6000g 1 minute
  • Add a further 30 μl EB, spin again
  • Discard the column and retain the eluted DNA


Restriction digests

  • Digest in a 100 μl final volume
  • Initial DNA is 50 μl from the elution
  • Add 10 μl Buffer 2
  • Add 1 μl BSA
  • Add 35 μl DI water
  • Add 2 μl EcoRI
  • Add 2 μl PstI
  • Digest 2 hours at 37°
  • Heat kill 20 minutes at 80°


Binding and removing uncut DNA and short ends to streptavidin-agarose

  • Use Pierce Streptavidin-agarose beads, Pierce 20349 [[1]]
  • These have high capacity, around 75 pmol/μl
  • Dispense 100 μl of the settled beads into a 15 ml centrifuge tube.
  • Add 10 ml of binding buffer, resuspending the beads
  • Wash for 30 minutes at room temperature with agitation
  • Centrifuge at 6000g for 1 minute
  • Discard the supernatent
  • Add 2 ml of binding buffer and resuspend the beads
  • Add the cut PCR product ( < 500 μl )
  • Bind for 30 minutes at room temperature with agitation
  • Centrifuge at 6000g for 1 minute
  • Pour off the supernatent into a clean 15 ml centifuge tube
  • Add 6 ml of cold ethanol
  • Freeze for 30 minutes at -80° to form a gel
  • Centrifuge at 12000g for 30 minutes to precipitate the recovered DNA
  • Wash the DNA pellet with 70% ethanol
  • A second 70% ethanol wash may be required to remove the high salt concentration present
  • Resuspend the purified DNA in TE


Binding buffer

  • 1 M NaCl
  • 20 mM Tris-HCl pH 7.5
  • 5 mM EDTA pH 8.0
  • 0.1% Tween-20 detergent


Construction Plasmid Biotin Primers

  • Primers amplify any Biobrick plasmid backbone
  • Order 50 nM, 5' biotin modification, HPLC purified
  • GTT TCT TCC TCT AGA AGC GGC CGC GAA TTC,Prefix-RB
  • GT TTC TTC TAC TAG TAG CGG CCG CTG CAG,Suffix-FB
  • Dilute to 30 pmol/μl with TE
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