Lidstrom:Gibson Assembly

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Design:
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Back to [[Lidstrom:Protocols]]
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- overlap by 40 bp
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==Design:==
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===Fragments===
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*overlap by 40 bp
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*efficiency
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===Oligo (primer) Design===
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*
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==PCR==
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==Assembly==
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== Transformation==
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*Electroporation can give much higher efficiency.  For this reason, the assembled product is usually electroporated.
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**rxn product is salty: can be a problem for electrocompetent cells.  Rob Egbert dilutes the competent cells when transforming with electroporation.  Not so much a problem when using chemically competent cells. 
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** Janet hasn't had a problem transforming 1 uL of product if a cuvette is new.  Older cuvettes arc more easily.
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*
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==Screening==
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* Colony PCR 1st
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* Sequence a few clones with primers that cover the "seams."
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==Misc: ==
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Misc:
 
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- rxn product is salty: can be a problem for electrocompetent cells.  Rob Egbert dilutes the competent cells when transforming with electroporation.  Not so much a problem when using chemically competent cells. 
 
- Electrocompetence is ~ 1000x more effective so is tempting.
- Electrocompetence is ~ 1000x more effective so is tempting.
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Revision as of 09:15, 2 October 2012

Back to Lidstrom:Protocols

Contents

Design:

Fragments

  • overlap by 40 bp
  • efficiency

Oligo (primer) Design

PCR

Assembly

Transformation

  • Electroporation can give much higher efficiency. For this reason, the assembled product is usually electroporated.
    • rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells.
    • Janet hasn't had a problem transforming 1 uL of product if a cuvette is new. Older cuvettes arc more easily.

Screening

  • Colony PCR 1st
  • Sequence a few clones with primers that cover the "seams."

Misc:

- Electrocompetence is ~ 1000x more effective so is tempting. -

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