Streptavidin purification of DNA fragments: Difference between revisions
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* 36 cycles | * 36 cycles | ||
** 95° 20 sec | ** 95° 20 sec | ||
** 68° 4:00 min | ** 62° 20 sec | ||
** 68° 4:00 min | |||
* final extension 68° 20 min | * final extension 68° 20 min | ||
Revision as of 09:35, 11 May 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
- 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
- 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 PCR product (300 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
- 400 ng of DNA
- 6 μl T4 DNA ligase buffer
- 1.5 μl T4 DNA ligase
- water to 60 μl
- Aliquot 4x 15 μl
- Add nothing to one
- Add 0.3 μl EcoRI to another
- Add 0.3 μl PstI to another
- Add 0.3 μl EcoRI and 0.3 μl PstI to a fourth
- Ligate 60 minutes at 16°
- Activate restriction enzymes at 37° for 30 minutes
- Heat kill ligase and enzymes at 80° for 20 minutes
- Run a gel
- Ligated band should show little single length fragment
- Ligated and single cut bands should show double length fragments
- Ligated and double cut bands 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 68°