Lidstrom:Gibson Assembly

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Back to [[Lidstrom:Protocols]]
Back to [[Lidstrom:Protocols]]
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== Janet's Tips ==
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'''Janet is developing a protocol [[Janet B. Matsen:Guide to Gibson Assembly |here]]'''
==Design:==
==Design:==
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*Electroporation can give much higher efficiency.  For this reason, the assembled product is usually electroporated.
*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.   
**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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** Janet electroporates 2 uL
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Current revision

Back to Lidstrom:Protocols

Contents

Janet's Tips

Janet is developing a protocol here

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 electroporates 2 uL

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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