Streptavidin purification of DNA fragments

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