Streptavidin purification of DNA fragments: Difference between revisions
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* Make primers for PCR reactions with a 5' biotin modification | * Make primers for PCR reactions with a 5' biotin modification | ||
** HPLC | ** 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 | ||
==PCR Reaction== | ==PCR Reaction== | ||
* 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 24: | Line 24: | ||
** 95° 20 sec | ** 95° 20 sec | ||
** 62° 20 sec | ** 62° 20 sec | ||
** 68° 4:00 min | ** 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 40: | Line 41: | ||
* Discard flow through, spin again at 12000g, 2 minutes to dry | * 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 | * 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 | * Discard the column and retain the eluted DNA | ||
* measure yield with the Nanodrop, expect 150-250 ng/μl in 45 μl | |||
==Restriction digests== | ==Restriction digests== | ||
* Digest in a 300 μl final volume | * Digest in a 300 μl final volume | ||
* Initial DNA is | * Initial DNA is 45 μl from the elution | ||
* Add 30 μl Buffer 2 | * Add 30 μl Buffer 2 | ||
* Add 3 μl BSA | * Add 3 μl BSA | ||
* Add | * Add 212 μl DI water | ||
* 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 μl 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/μl | ** These have high capacity, around 75 pmol/μl | ||
* Dispense 100 μl of the settled beads into a | * Dispense 100 μl of the settled beads into a 2 ml tube | ||
* Add | * 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 | * Wash for 30 minutes at room temperature with agitation | ||
* Centrifuge at | * Centrifuge at 8000g for 1 minute | ||
* Discard the supernatent | * Discard the supernatent | ||
* Add | * Add 300 μl of binding buffer and resuspend the beads | ||
* Add the cut PCR product ( | * Add the cut and adjusted PCR product (380 μl) | ||
* Bind | * Bind overnight at room temperature with agitation | ||
* Centrifuge at | * Centrifuge at 8000g for 1 minute in a Bio101 spin filter cartridge | ||
* | * Discard the filter | ||
* Add | * Add 1 μl of pellet-paint | ||
* Add 500 μl of isopropanol and mix | |||
* Freeze for 30 minutes at -80° to form a gel | * Freeze for 30 minutes at -80° to form a gel | ||
* Centrifuge at | * Centrifuge at 17000g for 30 minutes to precipitate the recovered DNA | ||
* Wash the DNA pellet with 70% ethanol | * Wash the DNA pellet with 70% ethanol | ||
* A | * 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== | ==Binding buffer== | ||
Line 78: | 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% | * 0.1% NP-40 detergent | ||
==Construction Plasmid Biotin Primers== | ==Construction Plasmid Biotin Primers== | ||
Line 88: | Line 121: | ||
* Dilute to 30 pmol/μl with TE | * Dilute to 30 pmol/μl with TE | ||
* Optimal annealing temperature seems to be about 62° | * Optimal annealing temperature seems to be about 62° | ||
==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/μl 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]] |
Latest revision as of 03:47, 23 February 2009
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