Streptavidin purification of DNA fragments

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(Binding and removing uncut DNA and short ends to streptavidin-agarose)
Current revision (06:47, 23 February 2009) (view source)
m (Restriction digests)
 
(21 intermediate revisions not shown.)
Line 2: Line 2:
* Make primers for PCR reactions with a 5' biotin modification
* 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
+
** 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
** Virtually all oligo manufacturers can supply 5' biotin
** An alternative is to make 5' amine primers and link biotin to the amine group
** An alternative is to make 5' amine primers and link biotin to the amine group
Line 9: Line 9:
* Normal PCR reaction conditions apply.
* Normal PCR reaction conditions apply.
* Approximately 1 pmol/μl biotinylated primer should be used.
* Approximately 1 pmol/μl biotinylated primer should be used.
 +
* For some reason, Phusion does not work (no product)
Line 22: Line 23:
* 36 cycles
* 36 cycles
** 95° 20 sec
** 95° 20 sec
-
** 68° 4:00 min (two step PCR with a 68° annealing)
+
** 62° 20 sec
 +
** 68° 4:00 min  
* final extension 68° 20 min
* final extension 68° 20 min
==Post PCR Cleanup==
==Post PCR Cleanup==
* Elimination of PCR enzymes and dNTPs is required prior to enzymatic cutting
* Elimination of PCR enzymes and dNTPs is required prior to enzymatic cutting
 +
* Add 2 μl 500 mM EDTA
* Add 1 μl Proteinase-K
* Add 1 μl Proteinase-K
* digest at 50° for 1 hour
* digest at 50° for 1 hour
Line 50: Line 53:
* Add 5 μl EcoRI
* Add 5 μl EcoRI
* Add 5 μl PstI
* Add 5 μl PstI
 +
* Add 1 μl [[DpnI]]
* Digest 2 hours at 37°
* Digest 2 hours at 37°
* Heat kill 20 minutes at 80°
* Heat kill 20 minutes at 80°
==Binding and removing uncut DNA and short ends to streptavidin-agarose==
==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<sup>++</sup> by adding 20 &mu;l of 500 mM EDTA
* Use Pierce Streptavidin-agarose beads, Pierce 20349 [[http://www.piercenet.com/files/0187as4.pdf]]
* Use Pierce Streptavidin-agarose beads, Pierce 20349 [[http://www.piercenet.com/files/0187as4.pdf]]
-
* These have high capacity, around 75 pmol/&mu;l
+
** These have high capacity, around 75 pmol/&mu;l
-
* Dispense 100 &mu;l of the settled beads into a 1.7 ml tube
+
* Dispense 100 &mu;l of the settled beads into a 2 ml tube
-
* Add 1.5 ml of binding buffer, resuspending the beads
+
* Add 1.7 ml of binding buffer, resuspending the beads
-
* Wash for 30 minutes at room temperature with agitation
+
* Wash 30 minutes at room temperature with agitation
* Centrifuge at 8000g for 1 minute
* Centrifuge at 8000g for 1 minute
* Discard the supernatent
* Discard the supernatent
-
* Add 1.5 ml of binding buffer, resuspending the beads
+
* Add 1.7 ml of binding buffer, resuspending the beads
* Wash for 30 minutes at room temperature with agitation
* Wash for 30 minutes at room temperature with agitation
* Centrifuge at 8000g for 1 minute
* Centrifuge at 8000g for 1 minute
* Discard the supernatent
* Discard the supernatent
* Add 300 &mu;l of binding buffer and resuspend the beads
* Add 300 &mu;l of binding buffer and resuspend the beads
-
* Add the cut PCR product (300 &mu;l)
+
* Add the cut and adjusted PCR product (380 &mu;l)
-
* Bind for 30 minutes at room temperature with agitation
+
* Bind overnight at room temperature with agitation
* Centrifuge at 8000g for 1 minute in a Bio101 spin filter cartridge
* Centrifuge at 8000g for 1 minute in a Bio101 spin filter cartridge
* Discard the filter
* Discard the filter
Line 77: Line 85:
* Resuspend the purified DNA in 50 &mu;l TE
* Resuspend the purified DNA in 50 &mu;l TE
* Quantitate the DNA
* 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==
==Binding buffer==
Line 82: Line 112:
* 20 mM Tris-HCl pH 7.5
* 20 mM Tris-HCl pH 7.5
* 5 mM EDTA pH 8.0
* 5 mM EDTA pH 8.0
-
* 0.1% Tween-20 detergent
+
* 0.1% NP-40 detergent
==Construction Plasmid Biotin Primers==
==Construction Plasmid Biotin Primers==
Line 91: Line 121:
* Dilute to 30 pmol/&mu;l with TE
* Dilute to 30 pmol/&mu;l with TE
* Optimal annealing temperature seems to be about 62&deg;
* Optimal annealing temperature seems to be about 62&deg;
 +
 +
==Ligation and Restriction enzyme buffers==
 +
 +
* T4 DNA Ligase Buffer
 +
** 50 mM Tris-HCl
 +
** 10 mM MgCl<sub>2</sub>
 +
**  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 MgCl<sub>2</sub>
 +
** pH 7.5
 +
** star activity with NaCl < 25 mM
 +
 +
* PstI (Buffer 3)
 +
** 50 mM Tris-HCl
 +
** 100 mM NaCl
 +
** 10 mM MgCl<sub>2</sub>
 +
** 1 mM DTT
 +
** low salt gives star activity
 +
 +
 +
[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]

Current revision

Contents

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 [[1]]
    • 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
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