Mesoplasma florum:Inverse PCR Transposon location

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Current revision (13:16, 23 February 2009) (view source)
 
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Materials
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{{back to protocols}}
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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==
* 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
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Ligation of cut ends at 5 ng/μl concentration (Hutchison99)
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==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
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Transposon detection PCR reaction 10 μl test volume
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==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
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Inverse PCR for transposon location identification
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==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%
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Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650
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==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
-
MboI cuts at GATC sites, insensitive to 5-me dCTP methylation (sensitive to methylation of A)
+
==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
-
MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231
+
==References==
-
 
+
Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650
-
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]]

Current revision

back to protocols

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.


Contents

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

  • 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%

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

References

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

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