Mesoplasma florum:Inverse PCR Transposon location: Difference between revisions
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Materials | ==Materials== | ||
* Genomic DNA from single colony transposon insertion event | * Genomic DNA from single colony transposon insertion event | ||
* MboI | * MboI | ||
| Line 11: | Line 11: | ||
* 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 | ||
| Line 19: | Line 19: | ||
* 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 | ||
| Line 26: | Line 26: | ||
* 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 | ||
| Line 45: | Line 45: | ||
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 | ||
Revision as of 12:57, 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%
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
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
