Streptavidin purification of DNA fragments

Biotin Primers

 * Make primers for PCR reactions with a 5' biotin modification
 * HPLC 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/&mu;l biotinylated primer should be used.
 * For some reason, Phusion does not work (no product)


 * 100 &mu;l reaction
 * 100 &mu;l PCR Supermix High Fidelity (Invitrogen)
 * 1.5 &mu;l Suffix-FB biotinylated primer (30 pmol/&mu;l)
 * 1.5 &mu;l Prefix-RB biotinylated primer (30 pmol/&mu;l)
 * 0.5 &mu;l diluted plasmid backbone template DNA (10 ng/&mu;l)


 * Cycle 36x
 * initial denature 95&deg; 2 min
 * 36 cycles
 * 95&deg; 20 sec
 * 62&deg; 20 sec
 * 68&deg; 4:00 min
 * final extension 68&deg; 20 min

Post PCR Cleanup

 * Elimination of PCR enzymes and dNTPs is required prior to enzymatic cutting
 * Add 2 μl 500 mM EDTA
 * Add 1 &mu;l Proteinase-K
 * digest at 50&deg; for 1 hour
 * heat kill Proteinase K at 80&deg; for 20 minutes
 * Add 5x (500 &mu;l) 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 &mu;l buffer PB, spin again
 * Discard flow through, add 750 &mu;l wash PE, spin again
 * Discard flow through, add 750 &mu;l 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 &mu;l EB heated to 50&deg;, spin at 8000g 1 minute
 * Add a further 30 &mu;l EB, spin again
 * Discard the column and retain the eluted DNA
 * measure yield with the Nanodrop, expect 150-250 ng/&mu;l in 45 &mu;l

Restriction digests

 * Digest in a 300 &mu;l final volume
 * Initial DNA is 45 &mu;l from the elution
 * Add 30 &mu;l Buffer 2
 * Add 3 &mu;l BSA
 * Add 212 &mu;l DI water
 * Add 5 &mu;l EcoRI
 * Add 5 &mu;l PstI
 * Add 1 &mu;l DpnI
 * Digest 2 hours at 37&deg;
 * Heat kill 20 minutes at 80&deg;

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

 * For binding uncut and short fragments, the salt concentration must be increased.
 * Adjust restriction digest to 1 M NaCl by adding 60 &mu;l of 5M NaCl
 * During the binding reaction, the exposed cut ends must be protected from exonucleases by removing the magnesium
 * Chelate Mg++ by adding 20 &mu;l of 500 mM EDTA
 * Use Pierce Streptavidin-agarose beads, Pierce 20349 []
 * These have high capacity, around 75 pmol/&mu;l
 * Dispense 100 &mu;l of the settled beads into a 2 ml tube
 * Add 1.7 ml of binding buffer, resuspending the beads
 * Wash 30 minutes at room temperature with agitation
 * Centrifuge at 8000g for 1 minute
 * Discard the supernatent
 * Add 1.7 ml of binding buffer, resuspending the beads
 * Wash for 30 minutes at room temperature with agitation
 * Centrifuge at 8000g for 1 minute
 * Discard the supernatent
 * Add 300 &mu;l of binding buffer and resuspend the beads
 * Add the cut and adjusted PCR product (380 &mu;l)
 * Bind overnight at room temperature with agitation
 * Centrifuge at 8000g for 1 minute in a Bio101 spin filter cartridge
 * Discard the filter
 * Add 1 &mu;l of pellet-paint
 * Add 500 &mu;l of isopropanol and mix
 * Freeze for 30 minutes at -80&deg; to form a gel
 * Centrifuge at 17000g for 30 minutes to precipitate the recovered DNA
 * Wash the DNA pellet with 70% ethanol
 * Resuspend the purified DNA in 50 &mu;l TE
 * Quantitate the DNA
 * expect about a 50% yield over the purified PCR product (3 to 6 &mu;g total, 50 to 150 ng/&mu;l)

Testing the purified DNA

 * Mix a master ligation mix containing
 * 250 ng of DNA
 * 7.5 &mu;l T4 DNA ligase buffer
 * water to 75 &mu;l
 * Set aside 15 &mu;l as a reference band A and add to it 1 &mu;l of 500 mM EDTA to remove magnesium
 * Add 0.3 &mu;l T4 DNA ligase
 * Restriction enzymes require some salt for activity
 * Adjust salt concentration to 25 mM by addition of 1.6 &mu;l of 1 M NaCl, mix
 * Aliquot 15 &mu;l samples to tubes B, C, D, and E
 * Add 0.3 &mu;l EcoRI to sample C
 * Add 0.3 &mu;l PstI to sample D
 * Add 0.3 &mu;l EcoRI and 0.3 &mu;l PstI to sample E
 * Ligate 60 minutes at 16&deg;
 * Cut for 10 minutes at 37&deg;
 * Heat kill for 20 minutes at 80&deg;
 * Run an 0.8% gel
 * Ligated band B should show little single length fragment and a high MW smear, with some double and quad length fragments
 * Ligated and single cut bands C and D should show double length fragments
 * Ligated and double cut band E should show single length fragments

Binding buffer

 * 1 M NaCl
 * 20 mM Tris-HCl pH 7.5
 * 5 mM EDTA pH 8.0
 * 0.1% NP-40 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/&mu;l with TE
 * Optimal annealing temperature seems to be about 62&deg;

Ligation and Restriction enzyme buffers

 * T4 DNA Ligase Buffer
 * 50 mM Tris-HCl
 * 10 mM MgCl2
 * 1 mM ATP
 * 10 mM DTT
 * 25 ng/&mu;l BSA
 * pH 7.5


 * EcoRI buffer
 * 100 mM Tris-HCl
 * 50 mM NaCl
 * 10 mM MgCl2
 * pH 7.5
 * star activity with NaCl < 25 mM


 * PstI (Buffer 3)
 * 50 mM Tris-HCl
 * 100 mM NaCl
 * 10 mM MgCl2
 * 1 mM DTT
 * low salt gives star activity