Difference between revisions of "Lidstrom:Gibson Assembly"

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(New page: Design: - overlap by 20 bp Misc: - rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not ...)
 
(Transformation)
 
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Design:
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Back to [[Lidstrom:Protocols]]
- overlap by 20 bp
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 +
== Janet's Tips ==
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'''Janet is developing a protocol [[Janet B. Matsen:Guide to Gibson Assembly |here]]'''
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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.
 +
**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 electroporates 2 uL
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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."
 +
 
 +
==Misc: ==
  
Misc:
 
- 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.
 
-
 
-

Latest revision as of 13:21, 22 March 2013

Back to Lidstrom:Protocols

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. -