Mesoplasma florum:Inverse PCR Transposon location

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
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 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]]

Revision as of 13:44, 8 September 2007

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

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

Personal tools