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
  • 3 μl Prefix-RB biotinylated primer
  • 1μl diluted plasmid backbone template DNA (less than 10 ng/μl)


  • Cycle 30x
  • initial denature 95° 2 min
  • 30 cycles
    • 95° 15 sec
    • 55° 15 sec
    • 68° 3 min (possibly longer for very large backbones)
  • final extension 68° 10 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
  • purify with a Qiagen column cleanup

Restriction digests

  • Normal restriction digests with less than 50 ng/μl DNA concentration.
  • For digestion with EcoRI and PstI, NEB Buffer 2 with BSA should be used.
  • Heat kill the enzymes for 20 minutes at 80C


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 (experiment with leaving this out)

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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