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
• 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
• Adding Proteinase-K followed by digestion at 50C for 1 hour is one approach
• Column purification may be easier and sufficient
• This purification will also eliminate some of the unused biotinylated primer
• verify correct product length and concentration by running a gel

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% NP-40 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