Streptavidin purification of DNA fragments
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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
- 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, spin again at 12000g, 2 minutes to dry
- Transfer column to a clean 1.7 ml tube, add 30 μl EB, spin at 6000g 1 minute
- Add a further 30 μl EB, spin again
- Discard the column and retain the eluted DNA
Restriction digests
- Digest in a 100 μl final volume
- Initial DNA is 50 μl from the elution
- Add 10 μl Buffer 2
- Add 1 μl BSA
- Add 35 μl DI water
- Add 2 μl EcoRI
- Add 2 μl PstI
- Digest 2 hours at 37°
- Heat kill 20 minutes at 80°
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% Tween-20 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