Mesoplasma florum:Inverse PCR Transposon location - Revision history

Torsten Waldminghaus at 17:16, 23 February 2009

Torsten Waldminghaus — Mon, 23 Feb 2009 17:16:04 GMT

←Older revision		Revision as of 17:16, 23 February 2009
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		+	{{back to protocols}}
	We identify the location of the transposon insertion event by cutting the genomic DNA with a frequent cutter,		We identify the location of the transposon insertion event by cutting the genomic DNA with a frequent cutter,
	religating, and using outward directed primers from the transposon insertion to amplify across the religation junction.		religating, and using outward directed primers from the transposon insertion to amplify across the religation junction.

Tk at 20:02, 8 September 2007

Tk — Sat, 08 Sep 2007 20:02:40 GMT

←Older revision		Revision as of 20:02, 8 September 2007
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		+	We identify the location of the transposon insertion event by cutting the genomic DNA with a frequent cutter,
		+	religating, and using outward directed primers from the transposon insertion to amplify across the religation junction.
		+	Sequencing the resulting fragment identifies the insertion location.
		+
		+
		+	==Choice of Enzyme==
		+
		+	* Hutchison used DraI as a cutter, but this is a blunt cutter, making religation difficult
		+	* MboI cuts at GATC sites, and is insensitive to 5-me dCTP methylation (sensitive to methylation of A)
		+	* MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231
		+	* Expected fragment length = 1/ .00231 = 432 bp + length from primer site to end of transposon
		+	* Expected PCR fragment length is twice this length, or about 1Kbp
		+
	==Materials==		==Materials==
	* Genomic DNA from single colony transposon insertion event		* Genomic DNA from single colony transposon insertion event
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	* The sequence from the M13F(-47) and T7 Universal primers should be adjacent, and oriented in opposite directions on the genome		* The sequence from the M13F(-47) and T7 Universal primers should be adjacent, and oriented in opposite directions on the genome

-		+	==References==
	Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650		Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650

-	~~MboI cuts at GATC sites, insensitive to 5-me dCTP methylation (sensitive to methylation of A)~~
-
-	~~MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231~~
-
-	~~Expected fragment length = 1/ .00231 = 432 bp + length from primer site to end of transposon~~
-
-	~~Expected PCR fragment length is twice this length, or about 1Kbp~~

	[[category:Mesoplasma florum]][[category:protocol]]		[[category:Mesoplasma florum]][[category:protocol]]

Tk at 19:57, 8 September 2007

Tk — Sat, 08 Sep 2007 19:57:38 GMT

←Older revision		Revision as of 19:57, 8 September 2007
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-	Materials	+	==Materials==
	* Genomic DNA from single colony transposon insertion event		* Genomic DNA from single colony transposon insertion event
	* MboI		* MboI
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	* E-Gel 0.8%		* E-Gel 0.8%

-	Restriction digest of 500 ng of genomic DNA with MboI	+	==Restriction digest of 500 ng of genomic DNA with MboI==
	* Mix 0.5 μl (approx) genomic DNA in TE		* Mix 0.5 μl (approx) genomic DNA in TE
	* 5 μl NEB buffer 3		* 5 μl NEB buffer 3
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	* heat kill at 65° for 20 minutes		* heat kill at 65° for 20 minutes

-	Ligation of cut ends at 5 ng/μl concentration (Hutchison99)	+	==Ligation of cut ends at 5 ng/μl concentration (Hutchison99)==
	* 5 μl digested DNA		* 5 μl digested DNA
	* 1 μl T4 DNA ligase buffer		* 1 μl T4 DNA ligase buffer
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	* incubate 10 minutes at room temperature		* incubate 10 minutes at room temperature

-	Transposon detection PCR reaction 10 μl test volume	+	==Transposon detection PCR reaction 10 μl test volume==
	* 0.5 μl ligated DNA		* 0.5 μl ligated DNA
	* 0.3 μl ME primer		* 0.3 μl ME primer
	9.2 μl PCR supermix High Fidelity		9.2 μl PCR supermix High Fidelity

-	Inverse PCR for transposon location identification	+	==Inverse PCR for transposon location identification==
	* 0.5 μl ligated DNA		* 0.5 μl ligated DNA
	* 0.15 μl M13forward(-47) primer		* 0.15 μl M13forward(-47) primer
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	Detect with E-Gel 0.8%		Detect with E-Gel 0.8%
		+
		+	==Inverse PCR for sequencing transposon location==
		+	* 5 μl ligated DNA
		+	* 1.5 μl M13forward(-47) primer
		+	* 1.5 μl T7 universal primer
		+	* 92 μl PCR supermix high fidelity
		+	* Cycle as above
		+	* Prepare 1% agarose prep gel
		+	* add 20 μl of loading dye
		+	* Run gel
		+	* Cut the band
		+	* Prepare DNA the gel with Qiaex II kit
		+	* Quantitate DNA
		+	* Sequence with both M13forward(-47) primer and T7 universal primer
		+
		+	==Sequence analysis==
		+	* locate the MboI cut site (GATC) in the sequencing results
		+	* locate the end of the transposon sequence (ME end, reverse complemented here, agatgtgtataagagacag)
		+	* Identify the duplicated 9bp insertion site surrounding the insertion event
		+	* Locate the sequence from the ME end to the GATC cut site on the ''Mesoplasma florum'' genome
		+	* The sequence from the M13F(-47) and T7 Universal primers should be adjacent, and oriented in opposite directions on the genome
		+

	Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650		Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650

Tk at 18:44, 8 September 2007

Tk — Sat, 08 Sep 2007 18:44:44 GMT

←Older revision		Revision as of 18:44, 8 September 2007
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	Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650		Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650
		+
		+	MboI cuts at GATC sites, insensitive to 5-me dCTP methylation (sensitive to methylation of A)

	MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231		MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231

-	Expected fragment length = 432 bp + length from primer site to end of transposon	+	Expected fragment length = 1/ .00231 = 432 bp + length from primer site to end of transposon

	Expected PCR fragment length is twice this length, or about 1Kbp		Expected PCR fragment length is twice this length, or about 1Kbp

	[[category:Mesoplasma florum]][[category:protocol]]		[[category:Mesoplasma florum]][[category:protocol]]

Tk at 16:53, 8 September 2007

Tk — Sat, 08 Sep 2007 16:53:47 GMT

←Older revision		Revision as of 16:53, 8 September 2007
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	MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231		MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231
		+
	Expected fragment length = 432 bp + length from primer site to end of transposon		Expected fragment length = 432 bp + length from primer site to end of transposon
		+
	Expected PCR fragment length is twice this length, or about 1Kbp		Expected PCR fragment length is twice this length, or about 1Kbp

	[[category:Mesoplasma florum]][[category:protocol]]		[[category:Mesoplasma florum]][[category:protocol]]

Tk at 16:53, 8 September 2007

Tk — Sat, 08 Sep 2007 16:53:16 GMT

←Older revision		Revision as of 16:53, 8 September 2007
Line 47:		Line 47:

	Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650		Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650
		+
		+	MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231
		+	Expected fragment length = 432 bp + length from primer site to end of transposon
		+	Expected PCR fragment length is twice this length, or about 1Kbp

	[[category:Mesoplasma florum]][[category:protocol]]		[[category:Mesoplasma florum]][[category:protocol]]

Tk at 20:07, 7 September 2007

Tk — Fri, 07 Sep 2007 20:07:59 GMT

←Older revision		Revision as of 20:07, 7 September 2007
Line 47:		Line 47:

	Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650		Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650
		+
		+	[[category:Mesoplasma florum]][[category:protocol]]

Tk: New page: Materials * Genomic DNA from single colony transposon insertion event * MboI * NEB buffer 3 10x * T4 DNA ligase buffer 10x * T4 DNA ligase * PCR supermix * M13forward(-47) primer * T7 uni...

Tk — Fri, 07 Sep 2007 20:07:06 GMT

New page: Materials * Genomic DNA from single colony transposon insertion event * MboI * NEB buffer 3 10x * T4 DNA ligase buffer 10x * T4 DNA ligase * PCR supermix * M13forward(-47) primer * T7 uni...

New page

Materials
* Genomic DNA from single colony transposon insertion event
* MboI
* NEB buffer 3 10x
* T4 DNA ligase buffer 10x
* T4 DNA ligase
* PCR supermix
* M13forward(-47) primer
* T7 universal primer
* ME primer
* E-Gel 0.8%

Restriction digest of 500 ng of genomic DNA with MboI
* Mix 0.5 μl (approx) genomic DNA in TE
* 5 μl NEB buffer 3
* 1 μl MboI
* 43.5 μl water
* incubate at 37° for 30 minutes
* heat kill at 65° for 20 minutes

Ligation of cut ends at 5 ng/μl concentration (Hutchison99)
* 5 μl digested DNA
* 1 μl T4 DNA ligase buffer
* 0.2 μl T4 DNA ligase
* 3.8 μl water
* incubate 10 minutes at room temperature

Transposon detection PCR reaction 10 μl test volume
* 0.5 μl ligated DNA
* 0.3 μl ME primer
9.2 μl PCR supermix High Fidelity

Inverse PCR for transposon location identification
* 0.5 μl ligated DNA
* 0.15 μl M13forward(-47) primer
* 0.15 μl T7 universal primer
* 9.2 μl PCR supermix high fidelity

* Cycle 5 minutes at 95° initial denturation
* 40 cycles of
** 94° 30 seconds
** 55° 30 seconds
** 64° 3 minutes
* 10 minutes 64° final extension

Detect with E-Gel 0.8%

Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650