EPF-Lausanne/15 September 2008

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==Sequencing==
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[[Image:epfl_gel-09-15-08.jpg|thumb|Agarose gel: Testing primers before sequencing]]
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[[Image:epfl_gel-09-15-08_2.jpg|thumb|Agarose gel: Annealing temp. reduced to 53°C for AB-3]]
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Before sending the plasmids for sequencing we test our primers by PCR.
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PCR on AB with:
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*pSB1A2 FW, I1466RV
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*AB1seqFW, I1466RV
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*AB2seqFW,LacImRV
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*R0071FW,pSB1A2RV
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PCR on CD with:
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*pSB1A2FW, C0040RV
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*CD1FW, C0040RV
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*R0040FW, BB3
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*R0040FW, pSB1A2RV
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'''Results:'''
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All results were as expected except for AB lane 3, i.e. AB2seqFW, LacImRV
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A new PCR is done for these primers, where the annealing temperature is reduced to 53°C.
 +
This turns out to work.

Latest revision as of 19:25, 28 October 2008

<<Previous - Back to Notebook - Next>>

Sequencing

Agarose gel: Testing primers before sequencing
Agarose gel: Annealing temp. reduced to 53°C for AB-3

Before sending the plasmids for sequencing we test our primers by PCR.

PCR on AB with:

  • pSB1A2 FW, I1466RV
  • AB1seqFW, I1466RV
  • AB2seqFW,LacImRV
  • R0071FW,pSB1A2RV

PCR on CD with:

  • pSB1A2FW, C0040RV
  • CD1FW, C0040RV
  • R0040FW, BB3
  • R0040FW, pSB1A2RV

Results:

All results were as expected except for AB lane 3, i.e. AB2seqFW, LacImRV

A new PCR is done for these primers, where the annealing temperature is reduced to 53°C. This turns out to work.

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