Mesoplasma florum:Inverse PCR Transposon location: Difference between revisions
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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]] |
Revision as of 09:53, 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 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