Mesoplasma florum:Inverse PCR Transposon location
来自 : 蚂蚁淘
- 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
- 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%
- 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
- 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
- 0.5 μl ligated DNA
- 0.3 μl ME primer
9.2 μl PCR supermix High Fidelity
- 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%
- 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
- 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
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