Streptavidin purification of DNA fragments

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(Construction Plasmid Biotin Primers)
(Binding and removing uncut DNA and short ends to streptavidin-agarose)
Line 56: Line 56:
==Binding and removing uncut DNA and short ends to streptavidin-agarose==
==Binding and removing uncut DNA and short ends to streptavidin-agarose==
 +
* Adjust restriction digest to 1 M NaCl by adding 60 μl of 5M NaCl
 +
* 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 1.7 ml tube
* Add 1.5 ml of binding buffer, resuspending the beads
* Add 1.5 ml of binding buffer, resuspending the beads
Line 68: Line 70:
* 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 for 30 minutes 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

Revision as of 01:04, 19 May 2007

Contents

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
  • 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 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

  • Adjust restriction digest to 1 M NaCl by adding 60 μl of 5M NaCl
  • 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 1.7 ml tube
  • Add 1.5 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 1.5 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 for 30 minutes 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

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
  • Add 1.5 μl T4 DNA ligase
  • Ligate the remaining 60 μl at 16° for 1 hour, heat kill ligase at 80° for 20 minutes
  • Remove 15 μl as ligation sample B
  • Adjust salt concentration to 50 mM by addition of 2.5 μl of 1 M NaCl
  • Aliquot 15 μl samples to tubes 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 samples C, D, E for 30 minutes at 37° and heat kill for 20 minutes at 80°
  • Run an 0.8% gel
    • Ligated band B should show little single length fragment
    • 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% 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
  • 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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