Streptavidin purification of DNA fragments - Revision history

Torsten Waldminghaus: /* Restriction digests */

2009-02-23T10:47:35Z

Restriction digests

←Older revision		Revision as of 10:47, 23 February 2009
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	* Add 5 μl EcoRI		* Add 5 μl EcoRI
	* Add 5 μl PstI		* Add 5 μl PstI
-	* Add 1 μl DpnI	+	* 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°

Tk: /* PCR Reaction */

2008-04-27T23:53:25Z

PCR Reaction

←Older revision		Revision as of 23:53, 27 April 2008
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	* 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)

Tk: /* Post PCR Cleanup */

2008-04-23T19:52:22Z

Post PCR Cleanup

←Older revision		Revision as of 19:52, 23 April 2008
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	==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

Tk: /* Binding buffer */

2007-07-25T22:52:21Z

Binding buffer

←Older revision		Revision as of 22:52, 25 July 2007
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	* 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% ~~Tween~~-20 detergent	+	* 0.1% NP-40 detergent

	==Construction Plasmid Biotin Primers==		==Construction Plasmid Biotin Primers==

Tk: /* Binding and removing uncut DNA and short ends to streptavidin-agarose */

2007-06-05T18:36:25Z

Binding and removing uncut DNA and short ends to streptavidin-agarose

←Older revision		Revision as of 18:36, 5 June 2007
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	* 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 ~~1.7~~ ml tube	+	* Dispense 100 μl of the settled beads into a 2 ml tube
-	* Add 1.5 ml of binding buffer, resuspending the beads	+	* Add 1.7 ml of binding buffer, resuspending the beads
	* Wash 30 minutes at room temperature with agitation		* Wash 30 minutes at room temperature with agitation
	* Centrifuge at 8000g for 1 minute		* Centrifuge at 8000g for 1 minute
	* Discard the supernatent		* Discard the supernatent
-	* Add 1.5 ml of binding buffer, resuspending the beads	+	* 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 8000g for 1 minute		* Centrifuge at 8000g for 1 minute

Tk: /* Biotin Primers */

2007-05-21T03:41:30Z

Biotin Primers

←Older revision		Revision as of 03:41, 21 May 2007
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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 ~~or PAGE~~ purification of these primers is desirable to eliminate short primers and ones without a biotin tag	+	** 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

Tk: /* Testing the purified DNA */

2007-05-21T02:22:09Z

Testing the purified DNA

←Older revision		Revision as of 02:22, 21 May 2007
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	** 7.5 μl T4 DNA ligase buffer		** 7.5 μl T4 DNA ligase buffer
	** water to 75 μl		** water to 75 μl
-	* Set aside 15 μl as a reference band A	+	* Set aside 15 μl as a reference band A and add to it 1 μl of 500 mM EDTA to remove magnesium
-	* Add 1.5 μl T4 DNA ligase	+	* Add 0.3 μl T4 DNA ligase
-	* ~~Ligate the remaining 60 μl at 16°~~ for ~~1 hour, heat kill ligase at 80° for 20 minutes~~	+	* Restriction enzymes require some salt for activity
-	* ~~Remove 15 μl as ligation sample B~~	+	** Adjust salt concentration to 25 mM by addition of 1.6 μl of 1 M NaCl, mix
-	* Adjust salt concentration to 50 mM by addition of 2.5 μl of 1 M NaCl	+	* Aliquot 15 μl samples to tubes B, C, D, and E
-	* Aliquot 15 μl samples to tubes C, D, and E	+
	** Add 0.3 μl EcoRI to sample C		** Add 0.3 μl EcoRI to sample C
	** Add 0.3 μl PstI to sample D		** Add 0.3 μl PstI to sample D
	** Add 0.3 μl EcoRI and 0.3 μl PstI to sample E		** Add 0.3 μl EcoRI and 0.3 μl PstI to sample E
	* Ligate 60 minutes at 16°		* Ligate 60 minutes at 16°
-	* Cut ~~samples C, D, E~~ for 30 minutes at 37° ~~and heat~~ kill for 20 minutes at 80°	+	* Cut for 10 minutes at 37°
		+	* Heat kill for 20 minutes at 80°
	* Run an 0.8% gel		* Run an 0.8% gel
-	** Ligated band B should show little single length fragment	+	** 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 single cut bands C and D should show double length fragments
	** Ligated and double cut band E should show single length fragments		** Ligated and double cut band E should show single length fragments

Tk: /* Binding and removing uncut DNA and short ends to streptavidin-agarose */

2007-05-21T02:07:54Z

Binding and removing uncut DNA and short ends to streptavidin-agarose

←Older revision		Revision as of 02:07, 21 May 2007
Line 56:		Line 56:

	==Binding and removing uncut DNA and short ends to streptavidin-agarose==		==Binding and removing uncut DNA and short ends to streptavidin-agarose==
-	* Adjust restriction digest to 1 M NaCl by adding 60 μl of 5M NaCl	+	* For binding uncut and short fragments, the salt concentration must be increased.
-	* Chelate Mg<sup>++</sup> by adding 20 μl of 500 mM EDTA	+	** 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 1.7 ml tube		* Dispense 100 μl of the settled beads into a 1.7 ml tube
	* Add 1.5 ml of binding buffer, resuspending the beads		* Add 1.5 ml of binding buffer, resuspending the beads
-	* Wash ~~for~~ 30 minutes at room temperature with agitation	+	* Wash 30 minutes at room temperature with agitation
	* Centrifuge at 8000g for 1 minute		* Centrifuge at 8000g for 1 minute
	* Discard the supernatent		* Discard the supernatent
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	* Add 300 μl of binding buffer and resuspend the beads		* Add 300 μl of binding buffer and resuspend the beads
	* Add the cut and adjusted PCR product (380 μl)		* Add the cut and adjusted PCR product (380 μl)
-	* Bind ~~for 30 minutes~~ at room temperature with agitation	+	* Bind overnight at room temperature with agitation
	* Centrifuge at 8000g for 1 minute in a Bio101 spin filter cartridge		* Centrifuge at 8000g for 1 minute in a Bio101 spin filter cartridge
	* Discard the filter		* Discard the filter
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	* Resuspend the purified DNA in 50 μl TE		* Resuspend the purified DNA in 50 μl TE
	* Quantitate the DNA		* 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==		==Testing the purified DNA==

Tk: /* Binding and removing uncut DNA and short ends to streptavidin-agarose */

2007-05-19T05:04:10Z

Binding and removing uncut DNA and short ends to streptavidin-agarose

←Older revision		Revision as of 05:04, 19 May 2007
Line 56:		Line 56:

	==Binding and removing uncut DNA and short ends to streptavidin-agarose==		==Binding and removing uncut DNA and short ends to streptavidin-agarose==
		+	* Adjust restriction digest to 1 M NaCl by adding 60 μl of 5M NaCl
		+	* 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 1.7 ml tube		* Dispense 100 μl of the settled beads into a 1.7 ml tube
	* Add 1.5 ml of binding buffer, resuspending the beads		* Add 1.5 ml of binding buffer, resuspending the beads
Line 68:		Line 70:
	* Discard the supernatent		* Discard the supernatent
	* Add 300 μl of binding buffer and resuspend the beads		* Add 300 μl of binding buffer and resuspend the beads
-	* Add the cut PCR product (~~300~~ μl)	+	* Add the cut and adjusted PCR product (380 μl)
	* Bind for 30 minutes at room temperature with agitation		* Bind for 30 minutes at room temperature with agitation
	* Centrifuge at 8000g for 1 minute in a Bio101 spin filter cartridge		* Centrifuge at 8000g for 1 minute in a Bio101 spin filter cartridge

Tk: /* Construction Plasmid Biotin Primers */

2007-05-13T00:41:23Z

Construction Plasmid Biotin Primers

←Older revision		Revision as of 00:41, 13 May 2007
Line 114:		Line 114:
	* 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]]		[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]