Mesoplasma florum:Transposome construction - Revision history

Kelly Drinkwater: added to category:protocol to make easier to find

2009-08-13T14:58:49Z

added to category:protocol to make easier to find

←Older revision		Revision as of 14:58, 13 August 2009
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-	[[category:Mesoplasma florum]]	+	[[category:Mesoplasma florum]][[category:protocol]]

Tk at 22:51, 18 February 2008

2008-02-18T22:51:11Z

←Older revision		Revision as of 22:51, 18 February 2008
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	==Transposome DNA construction using PCR and cutting==		==Transposome DNA construction using PCR and cutting==

-	* PCR with ext-ME primer using Phusion master mix. Final volume 200 μl~~, split 2x 100μl~~	+	* PCR with ext-ME primer using Phusion master mix. Final volume 200 μl
	** 100 μl 2x Phusion master mix		** 100 μl 2x Phusion master mix
	** 6 μl ext-ME primer (30 pM/μl) (gt ttc ttc agc tgt ctc tta tac aca tct)		** 6 μl ext-ME primer (30 pM/μl) (gt ttc ttc agc tgt ctc tta tac aca tct)
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	==Transposome DNA construction using PCR==		==Transposome DNA construction using PCR==

-	* PCR with ME0 primer using Phusion master mix. Final volume 200 μl	+	* PCR with ME0 primer using Phusion master mix. Final volume 200 μl, split 2x 100μl
	** 100 μl 2x Phusion master mix		** 100 μl 2x Phusion master mix
	** 6 μl ME0 primer (30 pM/μl) (phos- c tgt ctc tta tac aca tct)		** 6 μl ME0 primer (30 pM/μl) (phos- c tgt ctc tta tac aca tct)
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	** 93 μl water		** 93 μl water
	** Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m		** Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
-	* Add ~~1 ml~~ Qiagen buffer PB	+	* Add 2x 500 μl Qiagen buffer PB
	* Bind to standard Qiagen column, flowing past the column twice		* Bind to standard Qiagen column, flowing past the column twice
		+	* Wash once with 500 μl buffer PB
	* Wash 2x with 750μl buffer PE, spin after emptying.		* Wash 2x with 750μl buffer PE, spin after emptying.
-	* Elute with ~~100 and~~ 50 μl TE	+	* Elute with 2x 50 μl TE
-	* vacuum evaporate, resuspend in 10 μl TE	+	* vacuum evaporate, resuspend in 20 μl TE
-	* Measure concentration of the resulting DNA, expect 1 μg/μl	+	* Measure concentration of the resulting DNA, expect 500 ng/μl
-	* Dilute ~~0.5~~ μl into 20 μl, run a gel to verify correct size (TT01 is 2478 bp)	+	* Dilute to 100 ng/μl
		+	* Dilute 1 μl into 20 μl, run a gel to verify correct size (TT01 is 2478 bp)

Tk: /* Transposome DNA construction using PCR and cutting */

2008-02-18T22:47:12Z

Transposome DNA construction using PCR and cutting

←Older revision		Revision as of 22:47, 18 February 2008
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	==Transposome DNA construction using PCR and cutting==		==Transposome DNA construction using PCR and cutting==

-	* PCR with ext-ME primer using Phusion master mix. Final volume 200 μl	+	* PCR with ext-ME primer using Phusion master mix. Final volume 200 μl, split 2x 100μl
	** 100 μl 2x Phusion master mix		** 100 μl 2x Phusion master mix
	** 6 μl ext-ME primer (30 pM/μl) (gt ttc ttc agc tgt ctc tta tac aca tct)		** 6 μl ext-ME primer (30 pM/μl) (gt ttc ttc agc tgt ctc tta tac aca tct)
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	** 93 μl water		** 93 μl water
	** Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m		** Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
-	* Add ~~1 ml~~ Qiagen buffer PB	+	* Add 2x 500 μl Qiagen buffer PB
	* Bind to Qiaquick column 2x 600 μl, flowing past the column twice		* Bind to Qiaquick column 2x 600 μl, flowing past the column twice
-	* Wash 2x with 750μl buffer PE, spin after emptying.	+	* Wash once with 500 μl buffer PB
-	* Elute with 50 μl buffer EB into a clean tube.	+	* Wash 2x with 750μl buffer PE, spin at high speed for 5 minutes after emptying.
		+	* Elute with 2x 50 μl buffer EB into a clean tube.
	* Add 10 μl NEB buffer 2		* Add 10 μl NEB buffer 2
	* Add 37 μl water		* Add 37 μl water
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	* Elute with 20 μl TE		* Elute with 20 μl TE
	* Measure concentration of the resulting DNA		* Measure concentration of the resulting DNA
-

	==Transposome DNA construction using PCR==		==Transposome DNA construction using PCR==

Tk: /* Final transposome construction */

2008-02-18T20:06:58Z

Final transposome construction

←Older revision		Revision as of 20:06, 18 February 2008
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	==Final transposome construction==		==Final transposome construction==
-	* 1 μL ~~transposome~~ DNA, ~~1 μg~~/μL	+	* 1 μL transposon DNA, with phosphorylated ME ends, 100 ng/μL
	* 2 μL EZ-Transposase (Epicentre) [http://epibio.com/item.asp?ID=292]		* 2 μL EZ-Transposase (Epicentre) [http://epibio.com/item.asp?ID=292]
	* 1 μL glycerol		* 1 μL glycerol

Tk at 00:55, 18 February 2008

2008-02-18T00:55:27Z

←Older revision		Revision as of 00:55, 18 February 2008
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	* hold at RT for 1/2 hour		* hold at RT for 1/2 hour
	* reported to age at 4C overnight to provide higher efficiency		* reported to age at 4C overnight to provide higher efficiency
		+
		+
		+	[[category:Mesoplasma florum]]

Tk at 20:12, 1 January 2008

2008-01-01T20:12:23Z

←Older revision		Revision as of 20:12, 1 January 2008
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		+	==Transposome DNA construction from plasmid cutting==
	* Cut the transposome out of the containing plasmid with PvuII enzyme, which cuts at the correct location at the mosaic end.		* Cut the transposome out of the containing plasmid with PvuII enzyme, which cuts at the correct location at the mosaic end.
	* Cut also with an enzyme which produces shorter fragments from the remaining plasmid backbone to make gel purification easier. Sau3AI is a good enzyme.		* Cut also with an enzyme which produces shorter fragments from the remaining plasmid backbone to make gel purification easier. Sau3AI is a good enzyme.
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	** Measure concentration and label the tube		** Measure concentration and label the tube

-	==Transposome construction using PCR and cutting==	+	==Transposome DNA construction using PCR and cutting==

	* PCR with ext-ME primer using Phusion master mix. Final volume 200 μl		* PCR with ext-ME primer using Phusion master mix. Final volume 200 μl
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-	==Transposome construction using PCR==	+	==Transposome DNA construction using PCR==

	* PCR with ME0 primer using Phusion master mix. Final volume 200 μl		* PCR with ME0 primer using Phusion master mix. Final volume 200 μl
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	* Measure concentration of the resulting DNA, expect 1 μg/μl		* Measure concentration of the resulting DNA, expect 1 μg/μl
	* Dilute 0.5 μl into 20 μl, run a gel to verify correct size (TT01 is 2478 bp)		* Dilute 0.5 μl into 20 μl, run a gel to verify correct size (TT01 is 2478 bp)
		+
		+
		+	==Final transposome construction==
		+	* 1 μL transposome DNA, 1 μg/μL
		+	* 2 μL EZ-Transposase (Epicentre) [http://epibio.com/item.asp?ID=292]
		+	* 1 μL glycerol
		+	* hold at RT for 1/2 hour
		+	* reported to age at 4C overnight to provide higher efficiency

Tk: /* Transposome construction using PCR */

2007-12-31T20:57:12Z

Transposome construction using PCR

←Older revision		Revision as of 20:57, 31 December 2007
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	* Elute with 100 and 50 μl TE		* Elute with 100 and 50 μl TE
	* vacuum evaporate, resuspend in 10 μl TE		* vacuum evaporate, resuspend in 10 μl TE
-	* Measure concentration of the resulting DNA	+	* Measure concentration of the resulting DNA, expect 1 μg/μl
		+	* Dilute 0.5 μl into 20 μl, run a gel to verify correct size (TT01 is 2478 bp)

Tk: /* Transposome construction using PCR */

2007-12-31T20:55:55Z

Transposome construction using PCR

←Older revision		Revision as of 20:55, 31 December 2007
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	** Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m		** Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
	* Add 1 ml Qiagen buffer PB		* Add 1 ml Qiagen buffer PB
-	* Bind to ~~2x minelute~~ column ~~600 μl~~, flowing past the column twice	+	* Bind to standard Qiagen column, flowing past the column twice
	* Wash 2x with 750μl buffer PE, spin after emptying.		* Wash 2x with 750μl buffer PE, spin after emptying.
-	* Elute with ~~2x 10~~ μl TE, ~~combine~~	+	* Elute with 100 and 50 μl TE
		+	* vacuum evaporate, resuspend in 10 μl TE
	* Measure concentration of the resulting DNA		* Measure concentration of the resulting DNA

Tk: /* Transposome construction using PCR */

2007-12-29T22:38:10Z

Transposome construction using PCR

←Older revision		Revision as of 22:38, 29 December 2007
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	** Measure concentration and label the tube		** Measure concentration and label the tube

-	==Transposome construction using PCR==	+	==Transposome construction using PCR and cutting==

	* PCR with ext-ME primer using Phusion master mix. Final volume 200 μl		* PCR with ext-ME primer using Phusion master mix. Final volume 200 μl
	** 100 μl 2x Phusion master mix		** 100 μl 2x Phusion master mix
-	** 6 μl ext-ME primer (30 pg/μl) (gt ttc ttc agc tgt ctc tta tac aca tct)	+	** 6 μl ext-ME primer (30 pM/μl) (gt ttc ttc agc tgt ctc tta tac aca tct)
	** 1 μl transposon template (10 ng/μl)		** 1 μl transposon template (10 ng/μl)
	** 93 μl water		** 93 μl water
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	* Spin dry		* Spin dry
	* Elute with 20 μl TE		* Elute with 20 μl TE
		+	* Measure concentration of the resulting DNA
		+
		+
		+	==Transposome construction using PCR==
		+
		+	* PCR with ME0 primer using Phusion master mix. Final volume 200 μl
		+	** 100 μl 2x Phusion master mix
		+	** 6 μl ME0 primer (30 pM/μl) (phos- c tgt ctc tta tac aca tct)
		+	** 1 μl transposon template (10 ng/μl)
		+	** 93 μl water
		+	** Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m
		+	* Add 1 ml Qiagen buffer PB
		+	* Bind to 2x minelute column 600 μl, flowing past the column twice
		+	* Wash 2x with 750μl buffer PE, spin after emptying.
		+	* Elute with 2x 10 μl TE, combine
	* Measure concentration of the resulting DNA		* Measure concentration of the resulting DNA

Tk: /* Transposome construction using PCR */

2007-11-08T23:19:29Z

Transposome construction using PCR

←Older revision		Revision as of 23:19, 8 November 2007
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	** 100 μl 2x Phusion master mix		** 100 μl 2x Phusion master mix
	** 6 μl ext-ME primer (30 pg/μl) (gt ttc ttc agc tgt ctc tta tac aca tct)		** 6 μl ext-ME primer (30 pg/μl) (gt ttc ttc agc tgt ctc tta tac aca tct)
-	** 1 μl transposon template (10 ng/μl~~) (gt ttc ttc agc tgt ctc tta tac aca tct~~)	+	** 1 μl transposon template (10 ng/μl)
	** 93 μl water		** 93 μl water
	** Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m		** Cycle 98/30s, 30x (98/15s, 55/15s, 72/45s) 72/5m