Streptavidin purification of DNA fragments-PDF

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/μl biotinylated primer should be used.
• For some reason, Phusion does not work (no product)
• 100 μl reaction
• 100 μl PCR Supermix High Fidelity (Invitrogen)
• 1.5 μl Suffix-FB biotinylated primer (30 pmol/μl)
• 1.5 μl Prefix-RB biotinylated primer (30 pmol/μl)
• 0.5 μl diluted plasmid backbone template DNA (10 ng/μl)
• Cycle 36x
• initial denature 95° 2 min
• 36 cycles
  • 95° 20 sec
  • 62° 20 sec
  • 68° 4:00 min
• final extension 68° 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 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, 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 heated to 50°, spin at 8000g 1 minute
• Add a further 30 μl EB, spin again
• Discard the column and retain the eluted DNA
• measure yield with the Nanodrop, expect 150-250 ng/μl in 45 μl

Restriction digests

• Digest in a 300 μl final volume
• Initial DNA is 45 μl from the elution
• Add 30 μl Buffer 2
• Add 3 μl BSA
• Add 212 μl DI water
• Add 5 μl EcoRI
• Add 5 μl PstI
• Add 1 μl DpnI
• Digest 2 hours at 37°
• Heat kill 20 minutes at 80°

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 μl 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 μl of 500 mM EDTA
• Use Pierce Streptavidin-agarose beads, Pierce 20349
  • These have high capacity, around 75 pmol/μl
• Dispense 100 μl 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 μl of binding buffer and resuspend the beads
• Add the cut and adjusted PCR product (380 μl)
• Bind overnight at room temperature with agitation
• Centrifuge at 8000g for 1 minute in a Bio101 spin filter cartridge
• Discard the filter
• Add 1 μl of pellet-paint
• Add 500 μl of isopropanol and mix
• Freeze for 30 minutes at -80° 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 μl TE
• Quantitate the DNA
  • expect about a 50% yield over the purified PCR product (3 to 6 μg total, 50 to 150 ng/μl)

Testing the purified DNA

• Mix a master ligation mix containing
  • 250 ng of DNA
  • 7.5 μl T4 DNA ligase buffer
  • water to 75 μl
• Set aside 15 μl as a reference band A and add to it 1 μl of 500 mM EDTA to remove magnesium
• Add 0.3 μl T4 DNA ligase
• Restriction enzymes require some salt for activity
  • Adjust salt concentration to 25 mM by addition of 1.6 μl of 1 M NaCl, mix
• Aliquot 15 μl samples to tubes B, C, D, and E
  • Add 0.3 μl EcoRI to sample C
  • Add 0.3 μl PstI to sample D
  • Add 0.3 μl EcoRI and 0.3 μl PstI to sample E
• Ligate 60 minutes at 16°
• Cut for 10 minutes at 37°
• Heat kill for 20 minutes at 80°
• 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/μl with TE
• Optimal annealing temperature seems to be about 62°

Ligation and Restriction enzyme buffers

• T4 DNA Ligase Buffer
  • 50 mM Tris-HCl
  • 10 mM MgCl₂
  • 1 mM ATP
  • 10 mM DTT
  • 25 ng/μl 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