Imperial College/29 September 2008

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==Wet Lab==
==Wet Lab==
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===PCR===
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*Today we carried out a PCR to create the EpsE integration parts. These integration parts integrate at the 5' and the 3' of the EpsE gene. The image below summaries the results of the PCR. The labels correspond to the following:
 +
**E5'-EpsE integration site at 5' end of the gene,
 +
**E3'-EpsE integration site at 3' end of the gene,
 +
**Pos-Positive control of AmyE 5' (577bp)
 +
**Neg- Negative control, no DNA template.
 +
 +
[[Image:PCR29.PNG|400px]]
 +
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*The following reaction conditions were used with the Pfu ultra enzyme:
 +
**1 cycle - 95<sup>o</sup>C for 30 secs,
 +
**10 cycles - 95<sup>o</sup>C for 30 sec, 50<sup>o</sup>C for 60 sec,68<sup>o</sup>C for 30 sec
 +
**20 cycles - 95<sup>o</sup>C for 30 sec, 60<sup>o</sup>C for 60 sec,68<sup>o</sup>C for 30 sec
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*1 cycle - 68<sup>o</sup>C for 5 mins

Revision as of 20:56, 29 September 2008

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Wet Lab

PCR

  • Today we carried out a PCR to create the EpsE integration parts. These integration parts integrate at the 5' and the 3' of the EpsE gene. The image below summaries the results of the PCR. The labels correspond to the following:
    • E5'-EpsE integration site at 5' end of the gene,
    • E3'-EpsE integration site at 3' end of the gene,
    • Pos-Positive control of AmyE 5' (577bp)
    • Neg- Negative control, no DNA template.

PCR29.PNG

  • The following reaction conditions were used with the Pfu ultra enzyme:
    • 1 cycle - 95oC for 30 secs,
    • 10 cycles - 95oC for 30 sec, 50oC for 60 sec,68oC for 30 sec
    • 20 cycles - 95oC for 30 sec, 60oC for 60 sec,68oC for 30 sec
  • 1 cycle - 68oC for 5 mins
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