Streptavidin purification of DNA fragments: Difference between revisions
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** 68° 3 min (possibly longer for very large backbones) | ** 68° 3 min (possibly longer for very large backbones) | ||
* final extension 68° 10 min | * final extension 68° 10 min | ||
==Post PCR Cleanup== | ==Post PCR Cleanup== | ||
Line 55: | Line 56: | ||
* Pour off the supernatent into a clean 15 ml centifuge tube | * Pour off the supernatent into a clean 15 ml centifuge tube | ||
* Add 6 ml of cold ethanol | * Add 6 ml of cold ethanol | ||
* Freeze for 30 minutes at -80 to form a gel | * Freeze for 30 minutes at -80° to form a gel | ||
* Centrifuge at 12000g for 30 minutes to precipitate the recovered DNA | * Centrifuge at 12000g for 30 minutes to precipitate the recovered DNA | ||
* Wash the DNA pellet with 70% ethanol | * Wash the DNA pellet with 70% ethanol | ||
Line 67: | Line 68: | ||
* 5 mM EDTA pH 8.0 | * 5 mM EDTA pH 8.0 | ||
* 0.1% NP-40 detergent (experiment with leaving this out) | * 0.1% NP-40 detergent (experiment with leaving this out) | ||
==Construction Plasmid Biotin Primers== | ==Construction Plasmid Biotin Primers== |
Revision as of 10:13, 9 April 2007
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
- Adding Proteinase-K followed by digestion at 50C for 1 hour is one approach
- Column purification may be easier and sufficient
- This purification will also eliminate some of the unused biotinylated primer
- verify correct product length and concentration by running a gel
Restriction digests
- Normal restriction digests with less than 50 ng/μl DNA concentration.
- For digestion with EcoRI and PstI, NEB Buffer 2 with BSA should be used.
- Heat kill the enzymes for 20 minutes at 80C
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% NP-40 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