Streptavidin purification of DNA fragments: Difference between revisions
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(New page: ==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 ...) |
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** 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. | ||
==Post PCR Cleanup== | ==Post PCR Cleanup== | ||
| Line 16: | Line 18: | ||
* This purification will also eliminate some of the unused biotinylated primer | * This purification will also eliminate some of the unused biotinylated primer | ||
* verify correct product length and concentration by running a gel | * verify correct product length and concentration by running a gel | ||
==Restriction digests== | ==Restriction digests== | ||
| Line 22: | Line 25: | ||
* Heat kill the enzymes for 20 minutes at 80C | * Heat kill the enzymes for 20 minutes at 80C | ||
==Binding and removing uncut DNA and short ends to | |||
==Binding and removing uncut DNA and short ends to streptavidin-agarose== | |||
* 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 | ||
| Line 31: | Line 35: | ||
* Discard the supernatent | * Discard the supernatent | ||
* Add 2 ml of binding buffer and resuspend the beads | * Add 2 ml of binding buffer and resuspend the beads | ||
* Add the cut PCR product | * Add the cut PCR product ( < 500 μl ) | ||
* Bind for 30 minutes at room temperature with agitation | * Bind for 30 minutes at room temperature with agitation | ||
* Centrifuge at 6000g for 1 minute | * Centrifuge at 6000g for 1 minute | ||
| Line 38: | Line 42: | ||
* 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 | ||
* Resuspend the DNA in TE | * 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 | |||
Revision as of 08:37, 20 March 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.
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
